Antibody/drug conjugates and methods of use

ABSTRACT

The invention relates to conjugates that bind to targets, methods of using conjugates that bind to targets and methods of treating undesirable or aberrant cell proliferation or hyperproliferative disorders, such as tumors, cancers, neoplasia and malignancies that express a target.

RELATED APPLICATIONS

This application is a continuation application of application Ser. No.14/067,819, filed Oct. 30, 2013, which claims the benefit of priority toapplication Ser. No. 61/720,257, filed Oct. 30, 2012, all of whichapplications are expressly incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 1, 2016, isnamed 026039-0448996SEQLIS.txt and is 331,478 bytes in size.

TECHNICAL FIELD

The invention relates to antibodies, and Heavy (H) chains and/or Light(L) chains of antibodies, and antibody fragments fused or conjugated todrugs, such as lytic peptide conjugates, methods of using conjugates,for example, in methods of treating undesirable or aberrant cellproliferation or hyperproliferative disorders, such as non-metastaticand metastatic neoplasias, cancers, tumors and malignancies that expresstargets that bind to such antibodies, and Heavy (H) chains and/or Light(L) chains of antibodies.

INTRODUCTION

The need to develop new therapeutics for treatment of primary tumors andmetastases is clearly evident when the five year survival rate of cancerpatients is considered: Only 10-40% for patients with lung, colorectal,breast and prostate cancer survive if diagnosed with distant metastaticdisease.

SUMMARY

The invention is based, at least in part on lytic domains fused to anantibody, lytic domains fused or conjugated to Heavy (H) chains and/orLight (L) chains of antibodies, and lytic domains fused or conjugated toantibody fragments, that bind to a target (e.g., Her2/neu, HumanEpidermal growth factor Receptor 2, also known as ErbB-2, or CD20). Suchfusions can also be referred to herein as antibody or polypeptideconjugates or fusion constructs. Contact of a cell with a lytic domainis believed to cause disruption of the cell membrane which results incell death. The antibody, or Heavy (H) chain and/or Light (L) chain ofan antibody that binds to the target allows the lytic domain to targetexpressing cells for destruction, including undesirable or aberrantproliferating cells or hyperproliferating cells, such as non-metastaticand metastatic neoplasias, cancers, tumors and malignancies, thatexpress the target to which the antibody or Heavy (H) chain and/or Light(L) chain binds. A number of non-metastatic and metastatic neoplastic,cancer, tumor and malignant cells overexpress targets, such as receptorsor ligands. For example, many non-metastatic and metastatic neoplasias,cancers, tumors and malignancies, express a receptor target (e.g.,Her2/neu or CD20) that can be used as a target of the antibody orpolypeptide conjugate or fusion construct.

Conjugates can be designed to bind to any cell or cell population thatexpresses a target of interest. An antibody, fragment thereof, or Heavy(H) chain and/or Light (L) chain of an antibody or fragment thereof,selected based upon the target to which it binds, can be linked to alytic domain. The resulting antibody or polypeptide conjugate or fusionconstruct can in turn reduce or inhibit proliferation of cells thatexpress or target, thereby reducing or inhibiting proliferation orgrowth of the target expressing cells.

Conjugates do not require cells to divide in order to kill the targetcells. Accordingly, conjugates are useful against dividing andnon-dividing cells.

In accordance with the invention, there are provided antibody andpolypeptide conjugates that include a lytic domain. In one embodiment,an antibody conjugate includes or consists of an antibody (or fragmentthereof) that binds to a target, linked to a lytic domain that includesor consists of an L- or D-amino acid sequence that includes a peptidesequence selected from for example, KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7),or an L- or D-amino acid sequence that includes a peptide selected fromKFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ IDNOs.:1-6) having one or more of the K residues substituted with any ofan F or L residue, one or more of the F residues substituted with any ofa K, A or L residue, or one or more of the A residues substituted withany of a K, F or L residue. In another embodiment, a polypeptideconjugate includes or consists of a Heavy (H) chain and/or Light (L)chain of an antibody or fragment thereof that binds to a target, linkedto a lytic domain that includes or consists of an L- or D-amino acidsequence selected from for example, KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7),or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6) havingone or more of the K residues substituted with any of an F or L residue,one or more of the F residues substituted with any of a K, A or Lresidue, or one or more of the A residues substituted with any of a K, For L residue.

In a more particular embodiment, an antibody or polypeptide conjugateincludes an antibody (or fragment thereof) or Heavy (H) chain and/orLight (L) chain of an antibody (e.g., trastuzumab or pertuzumab) orfragment thereof (trastuzumab or pertuzumab fragment) that binds toHer2/neu, linked to a lytic domain that includes or consists of an L- orD-amino acid sequence selected from, KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7),or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6) havingone or more of the K residues substituted with any of an F or L residue,one or more of the F residues substituted with any of a K, A or Lresidue, or one or more of the A residues substituted with any of a K, For L residue.

In accordance with the invention, there are also provided isolated andpurified conjugates that include or consist of an antibody (or fragmentthereof) or a Heavy (H) chain and/or Light (L) chain of an antibody orfragment thereof that binds to a target, and a second domain. In variousembodiments, a second domain includes or consists of a lytic domain:KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF or KFAKFAKKFAKFAKKFAKFA (SEQ IDNOs.:1-7). In additional embodiments, a second domain includes orconsists of: KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF or KFAKFAKKFAKFAKKFAKFA (SEQ IDNOs.:1-6) having one or more of the K residues substituted with any ofan F or L residue, one or more of the F residues substituted with any ofa K, A or L residue, or one or more of the A residues substituted withany of a K, F or L residue.

In accordance with the invention, there are further providedpolypeptides that include one or more lytic domains. In variousembodiments, a polypeptide includes or consists of a: 1) lytic domainlinked to the amino(NH₂)-terminus of an antibody Heavy (H) chain; 2) alytic domain linked to the amino(NH₂)-terminus of an antibody Light (L)chain; 3) a lytic domain linked to a carboxy(C)-terminus of the antibodyHeavy (H) chain; or 4) a lytic domain linked to a carboxy(C)-terminus ofthe antibody Light (L) chain. In additional various embodiments, apolypeptide includes or consists of a: 1) lytic domain linked to anamino(NH₂)-terminus and a lytic domain linked to a carboxy(C)-terminusof an antibody Heavy (H) chain; or 2) a lytic domain linked to theamino(NH₂)-terminus and a lytic domain linked to a carboxy(C)-terminusof an antibody Light (L) chain.

Specific non-limiting examples of targets include amino acid sequences(e.g., polypeptides, peptides, proteins), polysaccharides,oligosaccharides, carbohydrates, and lipids. Specific non-limitingclasses of targets include receptors and antigens.

Targets include receptors that bind to antigens, receptors or ligands,including hormones, growth factors, cluster of differentiation(collectively known as CD molecules or CD markers), hormone and growthfactor analogues, and fragments of hormones, hormone analogs, growthfactors, growth factor analogues, and fragments of growth factors andanalogues. Particular non-limiting examples of receptor targets includeHer2/neu (Human Epidermal growth factor Receptor 2, also known asErbB-2), luteinizing hormone releasing hormone receptor (LHRH-R),epidermal growth factor (EGF) receptor, folate, and growth hormone (GH)receptor. Particular non-limiting examples of CD domains include CD19,CD20, CD22, CD23, CD27, CD28, CD30, CD31, CD33, CD34, CD40, CD52, CD56,CD70, CD123, CD138, or CD154, and others.

Antigen targets include viral, bacterial, fungal and parasite antigens.Antigen targets also include tumor associated antigens (TAAs).

An antibody includes 2 Heavy (H) chains and 2 Light (L) chains as wellas antibody fragments. A polypeptide that includes or consists of aHeavy (H) chain and/or Light (L) chain of an antibody or fragment of aHeavy (H) chain or Light (L) chain can include a single H or L chain ora single H or L chain fragment, or a plurality (2, 3, 4 or more) ofHeavy (H) chains and/or Light (L) chains, or a plurality of fragments ofHeavy (H) chains and/or Light (L) chains. A polypeptide that includes aHeavy (H) chain and/or Light (L) chain of an antibody or fragment canbut is not required to include 2 Heavy (H) chains and 2 Light (L) chainsand therefore polypeptide conjugates as set forth herein can excludenative antibodies that comprise 2 Heavy (H) chains and 2 Light (L)chains.

An antibody or fragment thereof may be an oligomeric (higher order orvalent) forms, such as a trimer, tetramer, pentamer, hexamer, heptamer,and so forth, with other antibodies, fragments thereof, Heavy (H) chain,Light (L) chain, or polypeptides sequence distinct from an antibodyHeavy (H) or Light (L) chain. Antibodies include monoclonals andfragments of monoclonal antibodies. Antibodies include mammalian, human,humanized, primatized and chimeric sequences.

Amino acid sequences (e.g., polypeptides, peptides, proteins,antibodies, Heavy (H) chains, Light (L) chains, lytic domains, etc.)include or consist of natural (L-) or non-natural (e.g., D-) aminoacids. In particular aspects, an amino acid sequence has about 2 to 10,10 to 14, 15 to 20, (i.e., 15, 16, 17, 18, 19 or 20 amino acids), 10 to20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 250, 250 to300, or more amino acid residues. Full-length antibody Heavy (H) chains,Light (L) chains, are typically 90 to 130 amino acids in length, but maybe shorter, for example, comprise a variable Heavy (H) chain, or Light(L) chain sequence, comprising one, two, or three complementaritydetermining regions (CDRs) with or without framework regions. Lyticdomains are typically 10 to 14, 15 to 20, (i.e., 15, 16, 17, 18, 19 or20 amino acids), 10 to 20, 20 to 30, 30 to 40, or 40 to 50, but mayoptionally be longer (50 or more) or shorter (less than 10). An aminoacid sequence can include or consist of a linear or cyclic structure.

Conjugates that include lytic domains can have the lytic domain at anylocation of the antibody (or fragment thereof) or Heavy (H) chain orLight (L) chain of an antibody. Thus, a lytic domain can be positionedat any amino acid position (amino acid residue) of the antibody (orfragment thereof) or Heavy (H) chain or Light (L) chain of an antibody.In addition, a conjugate can include multiple lytic domains.Accordingly, one or more (e.g., two, three, four, five, six, seven,eight or more) lytic domains linked to the Heavy (H) chain or Light (L)chain.) lytic domains can be included in a conjugate of the invention.

Lytic domains can also be positioned at the C-terminus, theNH₂-terminus, or both the C-terminus and the NH₂-terminus of apolypeptide sequence. In particular embodiments, a conjugate has a lyticdomain positioned at either (or both) the NH₂-terminus or the C-terminusof the antibody (or fragment thereof) Heavy (H) chain or Light (L)chain, or Heavy (H) chain or Light (L) chain. In particular aspects, alytic domain is linked to the amino(NH₂)-terminus of the Heavy (H) chainor linked to the carboxy(C)-terminus of the Heavy (H) chain; a lyticdomain is linked to the amino(NH₂)-terminus of the Light (L) chain orlinked to the carboxy(C)-terminus of the Light (L) chain. In a conjugatewith a plurality of lytic domains, such domains can be linked to theHeavy (H) chain or Light (L) chain, to the amino(NH₂)-terminus of theHeavy (H) chain, to the carboxy(C)-terminus of the Heavy (H) chain, tothe amino(NH₂)-terminus of the Light (L) chain, or to thecarboxy(C)-terminus of the Light (L) chain. In more particular aspects,at least one of a plurality of lytic domains is linked to theamino(NH₂)-terminus of the Heavy (H) chain, and at least one is linkedto the amino(NH₂)-terminus of the Light (L) chain; at least one of thelytic domains is linked to the amino(NH₂)-terminus of the Heavy (H)chain, at least one of the lytic domains is linked to theamino(NH₂)-terminus of the Light (L) chain, and at least one of thelytic domains is linked to the carboxy(C)-terminus of the Heavy (H)chain or is linked to the carboxy(C)-terminus of the Light (L) chain;and at least one of a plurality of lytic domains is linked to theamino(NH₂)-terminus of the Heavy (H) chain, at least one of the lyticdomains is linked to the amino(NH₂)-terminus of the Light (L) chain, atleast one of the lytic domains is linked to the carboxy(C)-terminus ofthe Heavy (H) chain and at least one of the lytic domains is linked tothe carboxy(C)-terminus of the Light (L) chain.

In embodiments in which conjugates include a plurality of lytic domains,the lytic domains have an identical amino acid sequence, or have adifferent amino acid sequence. Accordingly a conjugate can include two,a third, fourth, fifth, sixth, seventh lytic domain, etc., any or all ofwhich may be identical or different from each other.

In particular embodiments, a lytic domain is joined to a Heavy (H) chainor Light (L) chain immediately after the last amino acid at theamino(NH₂)-terminus or the carboxy(C)-terminus of the Heavy (H) chain orthe Light (L) chain, for example, by a covalent (e.g., peptide ornonpeptide) bond thereby forming a continuous amino acid sequencebetween the lytic domain and the Heavy (H) chain or Light (L) chain. Inadditional embodiments, antibodies, Heavy (H) chains and Light (L)chains and lytic domains can be joined by a peptide or a non-peptidelinker or spacer. In particular aspects, antibodies, Heavy (H) chainsand Light (L) chains and lytic domains and lytic domains are joined by apeptide sequence having from about 1 to 25 amino acid residues, or arejoined by a linear carbon chain, such as C_(N) (where N=1-100 carbonatoms, e.g., C, CC, CCC, CCCC, CCCCC, CCCCCC, CCCCCCC, CCCCCCCC, etc.).In more particular aspects, antibodies, Heavy (H) chains and Light (L)chains and lytic domains and lytic domains are joined by a peptidesequence that includes or consist of one or more A, S or G amino acidresidues (e. g., a peptide sequence including or consisting of GSGGS(SEQ ID No.:8), ASAAS (SEQ ID NO.:9), GS, AF, FK, VK, FFK, FA, GSGRSA(SEQ ID NO.:10), RVRRSV (SEQ ID NO.:11), SS, Cit-V (Cit=Citrulline(H₂NC(O)NH(CH₂)₃CH(NH₂)CO₂H); Val=Valine), F-Cit (F=Phenylalanine,Cit=Citrulline).

Conjugates further include or consist of additional (e.g., non-lytic)domains. Thus, in various aspects, a conjugate includes a second, third,fourth, fifth, sixth, seventh domain, etc., which may be distinct fromone or more (or all) lytic domains included in the conjugate.

Conjugates include or consist of isolated and/or purified forms.Conjugates also include or consist of a formulation or a mixture. Suchformulations and mixtures include compositions, such as a mixture ofconjugate and a pharmaceutically acceptable carrier or excipientappropriate for use, administration to or in vivo contact with asubject, or a mixture of conjugate and an anti-cell proliferative orimmune stimulating agent.

Conjugates include or consist of a unit dosage form, such as a dosageform for use or administration to a subject. In one embodiment, aconjugate is a unit dosage to administer or in an amount effective to ortreat a subject having undesirable cell proliferation or ahyperproliferative disorder. In another embodiment, a conjugate is aunit dosage to administer or in an amount effective to treat a subjecthaving a neoplasia, tumor or cancer. In an additional embodiment, aconjugate is a unit dosage to administer or in an amount effective toreduce fertility of a subject.

Conjugates can be included within kits, optionally with instructions forpracticing a method or use of the invention. In one embodiment, a kitincludes a conjugate and instructions for reducing or inhibitingproliferation of a cell, reducing or inhibiting proliferation of ahyperproliferating cell, reducing or inhibiting proliferation of aneoplastic, tumor or cancer cell, treating a subject having ahyperproliferative disorder, treating a subject having a neoplasia,tumor or cancer, or reducing fertility of an animal.

There are also provided nucleic acids that encode conjugates. In variousembodiments, a nucleic acid sequence encodes a: 1) lytic domain linkedto the amino(NH₂)-terminus of the antibody Heavy (H) chain; 2) a lyticdomain linked to the amino(NH₂)-terminus of the antibody Light (L)chain; 3) a lytic domain linked to the carboxy(C)-terminus of theantibody Heavy (H) chain; or 4) a lytic domain linked to thecarboxy(C)-terminus of the antibody Light (L) chain, of an antibody orpolypeptide conjugate. In another embodiment, a nucleic acid sequenceencodes a: 1) lytic domain linked to the amino(NH₂)-terminus and a lyticdomain linked to the carboxy(C)-terminus of the antibody Heavy (H)chain; 2) a lytic domain linked to the amino(NH₂)-terminus and a lyticdomain linked to the carboxy(C)-terminus of the antibody Light (L)chain, of the antibody or polypeptide conjugate.

Nucleic acids can be included in a vector, such as an expression vectorthat when expressed in a cell encodes a conjugate. Host cells can betransformed with a nucleic acid (e.g., encoding all or a portion of aconjugate, e.g., a Heavy (H) chain and/or Light (L) chain sequencelinked to a lytic domain) in a vector, such that the cell expresses aconjugate encoded by the nucleic acid.

As disclosed herein, targets can be expressed in or on a cell. Cellsthat express a target to which a conjugate binds can be targeted forbinding by the conjugates of the invention. Accordingly, such cells canbe selectively targeted by selecting a conjugate that binds to a targetexpressed by the cells.

Non-limiting target expressing cells include, for example,hyperproliferative cells. Additional cells that express targets include,for example, breast, ovarian, uterine, cervical, stomach, lung, gastric,colon, bladder, glial, dermal (e.g., melanocytes), hematologic andendometrial cells.

Conjugates are useful for, among other things, reducing or inhibitingproliferation of a cell, reducing or inhibiting cell proliferation,reducing or inhibiting proliferation of a hyperproliferating cell,reducing or inhibiting proliferation of a neoplastic, tumor, cancer ormalignant cell and treating undesirable or aberrant cell proliferation,such as hyperproliferating cells or hyperproliferative disorders.Non-limiting examples of hyperproliferative disorders include benignhyperplasia, non-metastatic and metastatic neoplasias, cancers tumorsand malignancies.

In accordance with the invention, there are further provided methods ofreducing or inhibiting proliferation of a cell; methods of reducing orinhibiting cell proliferation; methods of reducing or inhibitingproliferation of a hyperproliferating cell; and methods of reducing orinhibiting proliferation of a neoplastic, tumor, cancer or malignantcell. In various embodiments, a method includes contacting a cell with aconjugate in an amount sufficient to reduce or inhibit proliferation ofthe cell; contacting a cell with a conjugate in an amount sufficient toreduce or inhibit cell proliferation; contacting a cell with a conjugatein an amount sufficient to reduce or inhibit proliferation of thehyperproliferating cell; and contacting a cell with a conjugate in anamount sufficient to reduce or inhibit proliferation of the neoplastic,tumor, cancer or malignant cell.

In accordance with the invention, there are moreover provided methods ofselectively reducing or inhibiting proliferation of a cell thatexpresses a target to which a conjugate binds; selectively reducing orinhibiting proliferation of a hyperproliferating cell that express atarget to which a conjugated binds; and selectively reducing orinhibiting proliferation of a neoplastic, tumor, cancer or malignantcell that expresses a target to which a conjugated binds. In variousembodiments, a method includes contacting a target expressing cell witha conjugate in an amount sufficient to reduce or inhibit proliferationof the cell; contacting a target expressing cell with the conjugate inan amount sufficient to reduce or inhibit proliferation of thehyperproliferating cell; and contacting a target expressing cell with aconjugate in an amount sufficient to reduce or inhibit proliferation ofthe neoplastic, tumor, cancer or malignant cell, wherein the conjugatebinds to a target expressed by the cell.

Exemplary cells to be targeted in accordance with the invention uses andmethods include cells that express any desired target. Such non-limitingcells therefore include, for example, breast, ovarian, uterine,cervical, stomach, lung, gastric, colon, bladder, glial, dermal (e.g.,melanocytes), hematologic and endometrial cells. More particularnon-limiting cells express a receptor, such as Her2/neu, an antigen(e.g., a tumor associated antigen) or a cluster of differentiationdomain (CD).

Methods performed include, among others, administering to or contactinga subject in need of inhibiting, reducing or preventing proliferation,survival, differentiation, death, or activity of a cell, such as ahyperproliferative cell or an undesirably proliferating cell. Exemplarysubjects include a subject having or at risk of having undesirable oraberrant cell proliferation; a subject having or at risk of having abenign hyperplasia; or a non-metastatic or metastatic neoplasia, cancer,tumor or malignancy (e.g., a solid or liquid tumor, in any of breast,ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder,glial, dermal (e.g., melanocytes), hematologic or endometrial cells).

In accordance with the invention, there are additionally provided usesand methods of treating a subject having a hyperproliferative disorderand uses and methods of treating a subject having a neoplasia, tumor,cancer or malignancy (metastatic, non-metastatic or benign). In variousembodiments, a use or method includes, administering to a subject anamount of the conjugate sufficient to treat the hyperproliferativedisorder; and administering to a subject an amount of the conjugatesufficient to reduce or inhibit proliferation of the neoplasia, tumor,cancer or malignancy.

Methods and uses include treating a subject having or at risk of havinga metastasis. For example, an amount of a conjugate effective to reduceor inhibit spread or dissemination of a tumor, cancer or neoplasia toother sites, locations or regions within the subject. In variousembodiments, a method or use reduces or inhibits metastasis of a primarytumor or cancer to one or more other sites, formation or establishmentof a metastasis at one or more other sites, locations or regions therebyreducing or inhibiting tumor or cancer relapse or tumor or cancerprogression. In further embodiments, a method or use reduces or inhibitsgrowth, proliferation, mobility or invasiveness of tumor or cancer cellsthat potentially or do develop metastases (e.g., disseminated tumorcells); reduces or inhibits formation or establishment of metastasesarising from a primary tumor or cancer to one or more other sites,locations or regions distinct from the primary tumor or cancer; reducesor inhibits growth or proliferation of a metastasis at one or more othersites, locations or regions distinct from the primary tumor or cancerafter the metastasis has formed or has been established; or reduces orinhibits formation or establishment of additional metastasis after themetastasis has been formed or established. In yet another embodiment, amethod or use reduces or inhibits relapse or progression of theneoplasia, tumor, cancer or malignancy.

In accordance with the invention, there are still further providedmethods and uses of reducing or inhibiting metastasis of a neoplasia,tumor, cancer or malignancy to other sites, or formation orestablishment of metastatic neoplasia, tumor, cancer or malignancy atother sites distal from a primary neoplasia, tumor, cancer ormalignancy. In various embodiments, a method includes administering to asubject an amount of the conjugate sufficient to reduce or inhibitmetastasis of the neoplasia, tumor, cancer or malignancy to other sites,or formation or establishment of metastatic neoplasia, tumor, cancer ormalignancy at other sites distal from the primary neoplasia, tumor,cancer or malignancy.

Neoplasia, tumor, cancer and malignancy treatable in accordance with theinvention therefore include metastatic, and non-metastatic or benignforms. Non-limiting examples include a solid cellular mass,hematopoietic cells, or a carcinoma, sarcoma (e.g. lymphosarcoma,liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma,rhabdomyosarcoma or fibrosarcoma), lymphoma, leukemia, adenoma,adenocarcinoma, melanoma, glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma,mesothelioma, reticuloendothelial, lymphatic or haematopoietic (e.g.,myeloma, lymphoma or leukemia) neoplasia, tumor, cancer or malignancy.

Neoplasia, tumor, cancer and malignancy treatable in accordance with theinvention can be present in or affect a lung (small cell lung ornon-small cell lung cancer), thyroid, head or neck, nasopharynx, throat,nose or sinuses, brain, spine, adrenal gland, pituitary gland, breast,ovarian, uterine, cervical, gastrointestinal (mouth, esophagus, stomach,duodenum, ileum, jejunum (small intestine), colon, rectum), lung,genito-urinary tract (uterus, ovary, cervix, endometrial, bladder,testicle, penis, prostate), glial, hematologic, endometrial, lymph,blood, muscle, dermal (e.g., melanocytes) or skin cell, kidney,pancreas, liver, bone, bone marrow, Wilm's tumors, biliary tract, B-ALL(B-cell lymphoblastic leukemia), stem cell, or hematologic neoplasia,tumor, cancer, or malignancy.

Methods and uses may be practiced alone, for example, in subjects thatare not candidates for other therapies (e.g., surgical resection,chemotherapy, immunotherapy, radiotherapy, thermal therapy, vaccination,etc.). Methods and uses may also be practiced with other treatments ortherapies (e.g., surgical resection, radiotherapy, ionizing or chemicalradiation therapy, chemotherapy, immunotherapy, local or regionalthermal (hyperthermia) therapy, or vaccination). Such treatments ortherapies can be administered prior to, substantially contemporaneouslywith (separately or in a mixture), or following administration of aconjugate. In one embodiment, a method or use includes administering ananti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer orimmune-enhancing treatment or therapy. In further embodiments, a methodor use includes administering an alkylating agent, anti-metabolite,plant extract, plant alkaloid, nitrosourea, hormone, nucleoside ornucleotide analog; cyclophosphamide, azathioprine, cyclosporin A,prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan,methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosinearabinoside, 5-azacytidine (5-AZC) and 5-azacytidine related compounds,bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine,lomustine, semustine, streptozotocin, hydroxyurea, cisplatin,carboplatin, oxiplatin, mitotane, procarbazine, dacarbazine, a taxane(e.g., taxol or paclitaxel), vinblastine, vincristine, doxorubicin ordibromomannitol, topoisomerase inhibitors, (irinotecan, topotecan,etoposide, teniposide), gemcitabine, pemetrexed etc.

Cell or immunotherapies include lymphocytes, plasma cells, macrophages,dendritic cells, T-cells, NK cells or B-cells; an antibody, a cellgrowth factor, a cell survival factor, a cell differentiative factor, acytokine or a chemokine. Additional agents that are applicable withconjugates in compostions, methods or uses of the invention includetargeted drugs or biologicals, such as antibodies (monoclonal) or smallmolecules.

Methods of the invention include providing a subject with a benefit. Inparticular embodiments, a method of treatment results in partial orcomplete destruction of the neoplastic, tumor, cancer or malignant cellmass, volume, size or numbers of cells, stimulating, inducing orincreasing neoplastic, tumor, cancer or malignant cell necrosis, lysisor apoptosis, reducing neoplasia, tumor, cancer or malignancy volumesize, cell mass, inhibiting or preventing progression or an increase inneoplasia, tumor, cancer or malignancy volume, mass, size or cellnumbers, or prolonging lifespan; results in reducing or decreasingseverity, duration or frequency of an adverse symptom or complicationassociated with or caused by the neoplasia, tumor, cancer or malignancy;results in reducing or decreasing pain, discomfort, nausea, weakness orlethargy; or results in increased energy, appetite, improved mobility orpsychological well being.

Subjects treatable in accordance with the methods include mammals. Inparticular embodiments, a subject is a human.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show cytotoxicity of recombinant1 scFv-CH₃ (nakedantibody), scFv-C_(H)3-GS-Phor18 and scFv-C_(H)3-GS-(KLAKLAK)₂KLAK (SEQID NO.:74) to Her2-neu receptor positive FIG. 1A) breast (SKBR-3); andFIG. 1B) ovarian (SKOV-3) cancer cell lines determined after 48 hours.

FIGS. 2A and 2B show median tumor volumes from mice treated with nakedmonoclonal antibody (MAb), FIG. 2A) MAb-Phor18 and FIG. 2B) recombinantscFv-C_(H)3 naked antibody and scFv-C_(H)3-Phor18 conjugates during thestudy period of 64 days in SKOV-3 xenografted mice in comparison withsaline injected mice.

FIG. 3 shows mean tumor weights from mice treated with MAb (naked),MAb-Phor18 and recombinantly scFv-C_(H)3 naked antibody andscFv-C_(H)3-Phor18 conjugates on day 64 in SKOV-3 xenografted mice incomparison with saline injected mice.

FIG. 4 shows mean body weights from mice treated with MAb (naked),MAb-Phor18 and recombinantly scFv-C_(H)3 naked antibody andscFv-C_(H)3-Phor18 conjugates during the study period of 64 days inSKOV-3 xenografted mice in comparison with saline injected mice.

FIG. 5 shows the pCMVTnT expression vector (Promega) used for ADC heavy(H) and light (L) chain expression.

FIG. 6 shows an anti-Phor18 immunoblot of ADCs produced: H1L1 (IgG1),H3L1 (C_(H)3-Phor18-IgG1), H1L3 (C_(L)-Phor18-IgG1), H2L2(Phor18-V_(L)-Phor18-V_(H)-IgG1) and H1L2 (Phor18-V_(L)-Phor18-IgG).

FIGS. 7A and 7B show a quality analysis of ADCs produced: antiHer2-antibody, H1L1 (naked), H1L3 (C_(L)-Phor18-IgG1) and H3L1(C_(H)3-Phor18-IgG1) using FIG. 7A) and anti-Phor18 probe; and FIG. 7B)anti-kappa light (L) chain probe, of reduced proteins.

FIG. 8 shows anti IgG and anti-Phor18 Immunoblot of ADCs produced: H1L3(C_(L)-Phor18-IgG1), H1L2 (Phor18-V_(L)-Phor18-IgG1 and H2L2(Phor18-V_(L)-Phor18-V_(H)-IgG1).

FIG. 9 shows effect of whole antibody CD20 and antibody CD20-Phor18conjugates—chemically linked—on Caspase 3/7 activities in Daudi cells.

FIGS. 10A and 10B show in vitro activity of recombinantly producedscFv-Phor18 compared to naked scFv in CD20 expressing Daudi cells. FIG.10A) Cell membrane integrity was determined after 2 h, and FIG. 10B)cell viability was determined after 24 h of incubation with each nakedand conjugated AB. Naked scFv did not disintegrate cell membranes orkilled the target cells, whereas scFv-Phor18 conjugates showed membranedisintegration and cell killing.

FIG. 11 shows in vitro caspase 3/7 activation measured for naked scFvand scFv-Phor18 conjugates. scFv-Phor 18 increased caspase 3/7activities in a concentration dependent manner. Staurosporine served ascontrol for caspase activation. Naked scFv did not detectably activatecaspases.

FIG. 12 illustrates expression vector for anti-CD20 ADC and nakedantibody expression used for transfection in a yeast system.

FIG. 13A-13D show in vitro activities of scFvFc andPhor18-V_(L)-scFvFc-C_(H)3-Phor18 in CD20 positive Daudi cell linesafter FIG. 13A) 4 and FIG. 13B) 24 hours, and in CD20 negative U937cells after FIG. 13C) 4 and FIG. 13D) 24 hours.

FIG. 14 illustrates expression vector for anti-CD20 ADC and nakedantibody expression.

DETAILED DESCRIPTION

The invention is based at least in part on a conjugate that includes aportion that binds to a target joined or fused to a second lytic domain.In a typical configuration, a conjugate includes a first target bindingdomain (e.g., an antibody, or a Heavy (H) chain and/or Light (L) chainof an antibody) and a second domain that includes a lytic portion, whichis/are directly or indirectly toxic to a cell, which can thereby reducecell proliferation or survival, or stimulate, induce, increase orenhance cell death, killing or apoptosis.

In accordance with the invention, there are provided conjugates thatinclude or consist of an antibody, or a Heavy (H) chain and/or Light (L)chain of an antibody, that bind to a target, and a second lytic or toxicdomain. In one embodiment, a conjugate includes an antibody or fragmentthereof and a lytic domain comprising or consisting of a 10-100 residueL- or D-amino acid sequence that includes a peptide sequence (selectedfrom amino acids such as Lysine=K, Phenylalanine=F and Alanine=A), forexample, KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA andKFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7). In another embodiment, aconjugate includes a Heavy (H) chain and/or Light (L) chain of anantibody and a lytic domain comprising or consisting of a 10-100 residueL- or D-amino acid sequence selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA andKFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7).

As used herein, the term “conjugate” or “fusion construct” andgrammatical variations thereof, means that the construct containsportions or sections that are derived from, obtained or isolated from,or are based upon or modeled after two different molecular entities thatare distinct from each other and do not typically exist together innature. That is, for example, a first portion of the conjugate includesor consists of an antibody or antibody fragment, or a Heavy (H) chainand/or Light (L) chain of an antibody, and a second portion of theconjugate includes or consists of a lytic portion or domain, each of thefirst and second portions/domains structurally distinct. A conjugate canalso be referred to as a “fusion construct,” wherein the conjugateincludes or consists of a of an antibody or antibody fragment, or aHeavy (H) chain and/or Light (L) chain of an antibody, that bind to atarget, and a second lytic domain or portion.

First domains and or second (lytic) domains of conjugates include orconsist of amino acid sequences (peptides, polypeptides, proteins,lectins), nucleic acids (DNA, RNA) and carbohydrates (saccharides,sialic acid, galactose, mannose, fucose, acetylneuraminic acid, etc.).The terms “amino acid sequence,” “protein,” “polypeptide” and “peptide”are used interchangeably herein to refer to two or more amino acids, or“residues,” covalently linked by an amide bond or equivalent. Amino acidsequences can be linked by non-natural and non-amide chemical bondsincluding, for example, those formed with glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides, orN,N′-dicyclohexylcarbodiimide (DCC). Non-amide bonds include, forexample, ketomethylene, aminomethylene, olefin, ether, thioether and thelike (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids.Peptides and Proteins, Vol. 7, pp 267-357 (1983), “Peptide and BackboneModifications,” Marcel Decker, N.Y.).

First and second (lytic) domains of a conjugate or fusion constructinclude L-amino acid sequences, D-amino acid sequences and amino acidsequences with mixtures of L-amino acids and D-amino acids. Conjugatesof amino acid sequences of first and second domains can be a linear or acyclic structure, and can be further conjugated to another distinctmoiety (e.g., third, fourth, fifth, sixth, seventh, etc. domains), formintra or intermolecular disulfide bonds, and also form higher ordermultimers or oligomers (trimers, tetramers, pentamers, hexamers,heptamers, etc.) with other conjugates having the same or differentantibody, Heavy (H) chain, Light (L) chain or lytic sequence, or withother entirely distinct molecules.

Exemplary lengths of conjugates are from about 10 to 15, 15 to 20, 20 to25, 25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 or moreamino acid residues in length. In particular embodiments, a first orsecond domain includes or consists of an amino acid sequence of about 1to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to80, 80 to 90, 90 to 100 or more residues. In more particularembodiments, a lytic domain includes or consists of a 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or moreresidue amino acid sequence, or a 10, 11, 12, 13, 15, 16, 17, 18, 19,20, 22, 23, 24, 25, 26, 27, 28, or more residue amino acid sequence.

Conjugate that includes or consists of a first portion antibody orantibody fragment, or a Heavy (H) chain and/or Light (L) chain of anantibody, where the first portion binds to a target (e.g., receptor),and a second portion that includes or consists of a lytic domain, thelytic domain can form an amphipathic alpha-helix. An amphipathicalpha-helix contains mostly hydrophilic amino acids on one side of thealpha-helix and the other side contains mostly hydrophobic amino acids.Since the alpha helix makes a complete turn for every 3.6 residues, theamino acid sequence of an amphipathic alpha helix alternates betweenhydrophilic and hydrophobic residues every 3 to 4 residues. A PNNPNNPrepeat pattern or motif is predicted to form an amphipathic alpha-helixwhere P represents a positively charged amino acid residue and N aneutral amino acid residue. A PNNPNNP repeat pattern provides a cationicbinding site for the lytic peptide to interact with a negatively chargedcell membrane and a hydrophobic site for membraneinteraction/penetration. Conjugates therefore include that with lyticdomains having one or more uninterrupted PNNPNNP repeat patterns ormotifs, or one or more interrupted PNNPNNP repeat patterns or motifs,which can form an amphipathic alpha-helix. For example, a 15 or 18residue amino acid sequence, such as KFAKFAKKFAKFAKK (SEQ ID NO.:1) andKFAKFAKKFAKFAKKFAK (SEQ ID NO.:4), has uninterrupted and interruptedPNNPNNP repeat motifs.

As disclosed herein, conjugates include antibodies and antibodyfragments that bind to a target. An “antibody” refers to any monoclonalor polyclonal immunoglobulin molecule, such as IgM, IgG, IgA, IgE, IgD,and any subclass thereof. Exemplary subclasses for IgG are IgG₁, IgG₂,IgG₃ and IgG₄ including engineered antibody subclasses and recombinantantibodies with various glycosylation patterns.

Conjugates also include a Heavy (H) chain or a Light (L) chain of anantibody, or a fragment of a Heavy (H) chain or a Light (L) chain of anantibody, that bind to a target. Such conjugates can have a plurality ofHeavy (H) chains and/or Light (L) chains of an antibody, or a fragmentof Heavy (H) chains and/or Light (L) chains of an antibody, that bind toa target.

Antibody fragments, and fragments of Heavy (H) chains and/or Light (L)chains of an antibody, include the hypervariable (target binding)region, or any or all of the complementarity determining regions (CDRs)or framework regions (FRs) within a Heavy (H) chain and/or Light (L)chain of an antibody, sufficient to confer target binding. Specificnon-limiting examples of antibody fragments include Fab, Fab′, F(ab′)₂,Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V_(L),V_(H), Camel Ig, V-NAR, VHH, trispecific (Fab₃), bispecific (Fab₂),diabody ((V_(L)-V_(H))₂ or (V_(H)-V_(L))₂), triabody (trivalent),tetrabody (tetravalent), minibody ((scFv-C_(H)3)₂), bispecificsingle-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv)₂-Fc, affibody(e.g., ZHer2-neu:2, ZHer2-neu:4 ZHer2-neu:7 ZHer2-neu:8), aptamer,avimer or nanobody.

Antibodies, Heavy (H) chains and Light (L) chains of an antibody, andfragments thereof, include those produced by or expressed on cells, suchas B cells, or synthesized or engineered to be produced by other cells,e.g., CHO cells. Such antibodies, Heavy (H) chains and Light (L) chainsof an antibody, and fragments thereof, include those with improvedcharacteristics, such as increased serum stability and/or half life invivo, PK, etc. (e.g., as described in Antibody Engineering Vol 1,Konterman R and Duebel S, eds., 2010, Springer, WO 2006/130834 andHorton et al., Cancer Res 68:8049 (2008)). Non-limiting mutations in theFc include, for example, I253A, H310A, H435R, H435Q, G236R, L328R,S239D, I1332E. Non-limiting mutations in IgG1 can be at residues 238,252, 253, 254, 255, 256, 265, 272, 289, 288, 303, 305, 307, 309, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413,415, 424, 433, 434, 435, 439 and/or 477 of the Fc region.

Antibodies, Heavy (H) chains and Light (L) chains of an antibody, andfragments thereof bind to a target. Specific non-limiting examples oftargets include amino acid sequences (e.g., polypeptides, peptides,proteins), polysaccharides, oligosaccharides, carbohydrates, and lipids.Specific non-limiting classes of targets include receptors and antigens.Such targets can be expressed by or on a cell (e.g., on the cellmembrane).

Targets include receptors that bind to hormones, growth factors, hormoneand growth factor analogues, and fragments of hormones, hormone analogs,growth factors, growth factor analogues, and fragments of growth factorsand analogues. Particular non-limiting examples of receptor targetsinclude Her2/neu (Human Epidermal growth factor Receptor 2, also knownas ErbB-2), luteinizing hormone releasing hormone receptor (LHRH-R),epidermal growth factor receptor (EGF-R), folate-, and growth hormone(GH) receptor. Further particular non-limiting examples of receptortargets include a tumor necrosis factor (TNF) family member receptor(e.g., TNF-alpha, TNF-beta (lymphtoxin, LT), TRAIL, Fas, LIGHT, or4-1BB) or oncofetoprotein (5T4). Additional particular non-limitingexamples of receptor targets include an immunoglobulin-like receptor(e.g., CD19, CD20, CD22, CD23, CD27, CD28, CD30, CD31, CD33, CD34, CD40,CD52, CD56, CD70, CD123, CD138, CD123, CD138, or CD154), or otherreceptors, (e.g., hormone receptor, a cytokine receptor, a growth factorreceptor, or a chemokine receptor).

Antigen targets include viral, bacterial, fungal and parasite antigens.Antigen targets also include tumor associated antigens (TAAs).Particular non-limiting examples of TAA targets include carcinoembryonicantigen (CEA), alpha-fetoprotein (AFP), prostate specific antigen (PSA),prostate specific membrane antigen (PSMA), CA-125 (epithelial ovariancancer), soluble Interleukin-2 (IL-2) receptor, RAGE-1, tyrosinase,MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1, glycoprotein (gp) 75, gp100,beta-catenin, PRAME, MUM-1, ZFP161, Ubiquilin-1, HOX-B6, YB-1,Osteonectin, ILF3, IGF-1, oncofetoprotein, luteinizing hormone releasinghormone receptor (LHRH-R), growth hormone receptor, phosphatidylserine,follicle stimulating hormone receptors, VGEF receptor, folate receptor,CD19, CD20, CD22, CD23, CD27, CD28, CD30, CD31, CD33, CD34, CD40, CD52,CD56, CD70, CD123, CD138, or CD154.

As disclosed herein, receptors, such as Her2/neu or CD20 are typicallyexpressed by or present on (e.g., a membrane receptor) or within a cell.Receptors, such as Her2/neu, may associate with the cell membranesurface or traverse the cell membrane. CD20 is typically notinternalized and is expressed on the surface of B cells and B-cellmalignancies. Receptors therefore include full length intact nativereceptors containing an extracellular, transmembrane or cytoplasmicportion, as well as truncated forms or fragments thereof (e.g., anextracellular, transmembrane or cytoplasmic portion or subsequence of areceptor, such as Her2/neu alone, or in combination). For example, asoluble receptor such as Her2/neu typically lacks a transmembrane regionand can optionally also lack all or a part of the native extracellularor cytoplasmic region (if present in native receptor, e.g., Her2/neu).Such truncated forms and fragments can retain at least partial bindingto a conjugate.

Exemplary antibodies, Heavy (H) chains, Light (L) chains, and fragmentsthereof include those that bind to epitopes present on receptors.Exemplary Her2/neu epitopes to which antibodies, Heavy (H) chains orLight (L) chains of an antibody, or fragments thereof bind, includeHER-2 (p5-13) A2, HER-2 (p8-16) A24, HER-2 (p48-56) A2, HER-2 (p63-71)A24, HER-2 (p106-114) A2, HER-2 (p369-377) A2, A3, A26, HER-2 (p435-443)A2, HER-2 (p654-662) A2, HER-2 (p665-673) A2, HER-2 (p689-697) A2, HER-2(p754-762) A3, A11, A33, HER-2 (p773-782) A2, HER-2 (p780-788) A24,HER-2 (p785-794) A2, HER-2 (p789-797) A2, HER-2 (p799-807) A2, HER-2(p952-961) A2 and HER-2 (p1023-1032) A2 (the amino acid position numbersof Her2/neu epitope is referred to by a “p” followed by arabic numbers).

Exemplary antibodies that bind to Her2/neu include humanized anti-ErbB2antibodies huMAb4D1-1, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4,huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN™) asdescribed in U.S. Pat. No. 5,821,337; humanized 520C9 (WO 93/21319) andhumanized 2C4 (pertuzumab) as described in U.S. Pat. No. 7,097,840 andpertuzumab variants as described in US2009/0285837A1. Non-limitingrepresentative antibodies, Heavy (H) chains, Light (L) chains, andfragments thereof that bind to Her2/neu are set forth in Table A.

TABLE A (SEQ ID NOs.: 12-39) huMAb4D5-1: V_(L)(Light chain):1                 10                  20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G              50                  60                   70                  80K A P K L L I Y S A S F L E S G V P S R F S G S G S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                   20                  30                 40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                  70                 80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S R D D S K N T L Y L Q             90                100                 110                 120M N S L R A E D T A V Y Y C A R W G G D G F Y A M D V W G Q G T L V T V S ShuMAb4D5-2: V_(L)(Light chain):1                10                   20                 30                  40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G             50                  60                    70                  80K A P K L L I Y S A S F L E S G V P S R F S G S G S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                  20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                 80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D D S K N T L Y L Q             90                100                  110                 120M N S L R A E D T A V Y Y C A R W G G D G F Y A M D V W G Q G T L V T V S S huMAb4D5-3: V_(L)(Light chain):1                10                   20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G              50                  60                   70                   80K A P K L L I Y S A S F L E S G V P S R F S G S G S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                  20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D V W G Q G T L V T V S ShuMAb4D5-4: V_(L)(Light chain):1                10                   20                 30                  40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G              50                  60                   70                  80K A P K L L I Y S A S F L E S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                   20                  30                 40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T L Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D V W G Q G T L V T V S ShuMAb4D5-5: V_(L)(Light chain):1                10                   20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G             50                   60                   70                   80K A P K L L I Y S A S F L E S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                   20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P                50                 60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D V W G Q G T L V T V S ShuMAb4D5-6: V_(L)(Light chain):1                 10                  20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G             50                   60                   70                  80K A P K L L I Y S A S F L Y S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                  20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D V W G Q G T L V T V S ShuMAb4D5-7: V_(L)(Light chain):1                10                   20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G              50                  60                   70                  80K A P K L L I Y S A S F L E S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                  20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D Y W G Q G T L V T V S ShuMAb4D5-8: V_(L)(Light chain):1                10                  20                  30                  40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G             50                  60                    70                  80K A P K L L I Y S A S F L Y S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                   20                  30                 40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D Y W G Q G T L V T V S ShuMAb4D5: V_(L)(Light chain):1                10                   20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G              50                  60                  70                   80K A P K L L I Y S A S F L Y S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                   20                  30                 40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P               50                  60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D Y W G Q G T L V T V S SAntibody sequence permutations(U.S. Pat. No. 7,435,797 SQ 1 and 2))huMAb4D5: V_(L)(Light chain):1                 10                  20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G              50                  60                   70                  80K A P K L L I Y S A S F L Y S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                10                  20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P                50                 60                 70                  80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G D G F Y A M D Y W G Q G T L V T V S ShuMAb4D5-8(for VL: Q27, D28, N30, T31, A32, Y49, F53, Y55, R66 H91, Y92, T94; for VH: W95, D98, F100,Y100, Y102): V_(L)(Light chain): Claim 1 mutation1                10                   20                  30                 40D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V S S A V A W Y Q Q K P G              50                 60                  70                    80K A P K L L I D/W S A S F L Y S G V P S R F S G S R S G T D F T L T I S S L Q P E D F           90                  100A T Y Y C Q Q H Y T T P P T F G Q G T K V E I K V_(H)(heavy chain):1                 10                 20                   30                 40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P                50                 60                  70                 80G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q             90                 100                 110                 120M N S L R A E D T A V Y Y C S R W G G W G P K/L A M D Y W G Q G T L V T V S SPertuzumab Sequences (US2010/0015157A1) V_(L)(Light chain):1                10                   20                  30                  40D I Q M T Q S P S S L S A S V G D R V T I T C K A S Q D V S I G V A W Y Q Q K P G              50                 60                   70                   80K A P K L L I Y S A S Y R Y T G V P S R F S G S G S G T D F T L T I S S L Q P E D             90                  100                110                   120F A T Y Y C Q Q Y Y I Y P Y T F G Q G T K V E I K R T V A A P S V F I F P P S D E Q        130                   140                 150                   160L K S G T A S V V C L L N N N F Y P R E A K V Q W K V D N A L Q S G E N S Q E S             170                  180                190                 200V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G L S S           210 P V T K S F N R G E C V_(H)(heavy chain):1                10                   20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F T D Y T M D W V R Q A                 50                  60                  70                  80P G K G L E W V A D V N P N S G G S I Y N Q R F K G R F T L S V D R S K N T L Y L               90                 100                 110                  120Q M N S L R A E D T A V Y Y C A R N L G P S F Y F D Y W G Q G T L V T V S S A S T              130                  140                150                 160K G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S G           170                 180                 190                    200A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N       210                 220                230                   240H K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F L F P     250                 260                 270                 280P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H   290                300                 310                 320N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N   330                340                  350                  360K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C370                 380                 390                 400                410L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S                420                 430                  440K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P GPertuzumab variants (US2009/0285837 A1) V_(L)(Light chain):1                 10                  20                  30                  40V H S D I Q M T Q S P S S L S A S V G D R V T I T C K A S Q D V S I G V A W Y Q Q                  50                  60                  70                  80K P G K A P K L L I Y S A S Y R Y T G V P S R F S G S G S G T D F T L T I S S L Q P              90                  100                   110                 120E D F A T Y Y C Q Q Y Y I Y P Y T F G Q G T K V E I K R T V A A P S V F I F P P S D          130                140                 150                 160E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S          170                180                 190                200V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G L S S        210 P V T K S F N R G E C V_(H)(Heavy chain):1                 10                  20                  30                 40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F T D Y T M D W V R Q A                  50                  60                  70                  80P G K G L E W V A D V N P N S G G S I Y N Q R F K G R F T L S V D R S K N T L Y L                  90                 100                 110                  120Q M N S L R A E D T A V Y Y C A R N L G P S F Y F D Y W G Q G T L V T V S S A S                  130                 140                 150                 160T K G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S                170                 180                 190                 200G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V            210                 220                230                  240N H K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F L V           250                 260                 270                 280P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V          290                300                 310                 320H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S         330                  340                 350                 360N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T     370                  380                390                  400C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y    410                 420                   430                 440S K L T V D K S R W Q Q G N V F S C S V M H E A L N H Y T Q K S L S L S P G KPertuzumab Sequences: humanized 2C4, 7C2, 7F3, 7D3, 3E8, 4D5, 2H11, 3H4 (U.S. Pat. No.7,097,840) V_(L)(Light chain):1                 10                  20                  30                  40D I Q M T Q S P S S L S A S V G D R V T I T C K A S Q D V S I G V A W Y Q Q K P G              50                60                    70                   80K A P K L L I Y S A S Y R Y T G V P S R F S G S G S G T D F T L T I S S L Q P E D             90                  100                110                  120F A T Y Y C Q Q Y Y I Y P Y T F G Q G T K V E I K R T V A A P S V F I F P P S D E Q        130                   140                 150                   160L K S G T A S V V C L L N N N F Y P R E A K V Q W K V D N A L Q S G E N S Q E S             170                  180                190                 200V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G L S S           210 P V T K S F N R G E C V_(H)(heavy chain):1                 10                  20                  30                  40E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F T D Y T M D W V R Q A                 50                   60                 70                  80P G K G L E W V A D V N P N S G G S I Y N Q R F K G R F T L S V D R S K N T L Y L               90                 100                  110                 120Q M N S L R A E D T A V Y Y C A R N L G P S F Y F D Y W G Q G T L V T V S S A S T               130                 140                150                 160K G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S G           170                 180                 190                    200A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N       210                 220                230                   240H K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F L F P     250                 260                 270                280P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H   290                 300                 310                320N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N   330                340                  350                  360K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C370                 380                 390                 400                410L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S                420                 430                  440K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G

Additional anti-ErbB2 antibodies with various properties have beendescribed in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzieet al. Oncogene 4:543 (1989); Maier et al. Cancer Res. 51:5361 (1991);Bacus et al. Molecular Carcinogenesis 3:350 (1990); Stancovski et al.PNAS (USA) 88:8691 (1991); Bacus et al. Cancer Research 52:2580 (1992);Xu et al. Int. J Cancer 53:401-408 (1993); WO 94/00136; Kasprzyk et al.Cancer Research 52:2771 (1992); Hancock et al. Cancer Res. 51:4575(1991); Shawver et al. Cancer Res. 54:1367 (1994); Arteaga et al. CancerRes. 54:3758 (1994); Harwerth et al. J Biol Chem. 267:15160 (1992); U.S.Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099 (1997).Conjugates based on such antibodies, Heavy (H) chains and/or Light (L)chains of an antibody, and fragments thereof, are included in theinvention.

As disclosed herein, other scaffold like structures that bind to targets(e.g., receptors) can be included in an invention conjugate. Suchstructures include affibodies, aptamers, avimers and nanobodies.Conjugates that include such affibodies, aptamers, avimers andnanobodies are also included in the invention.

Exemplary Her2/neu binding affibodies, include ZHer2-neu:2, ZHer2-neu:4ZHer2-neu:7 ZHer2-neu: 8 and Fab63, which affibody sequences are inTable B.

TABLE B (SEQ ID NOs.: 40-47) Z_(wt)VDNKFNK EQQNAFYEILH LPNLNE EQRNAFIQSLKD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 4)VDNKFNK ELRQAYWEIQA LPNLNW TQSRAFIRSLYD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 7)VDNKFNK EPKTAYWEIVK LPNLNP EQRRAFIRSLYD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 24)VDNKFNK EPREAYWEIQR LPNLNN KQKAAFIRSLYD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 79)VDNKFNK EWMTAGKEIYR LPNLNG TQVRAFIQSLSD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 2)VDNKFNK EWVQAGSEIYN LPNLNR AQMRAFIRSLSD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 8)VDNKFNK EIKQAFHEIVR LPNLNA DQVRAFIYSLGD DPSQ SANLLAEAKKLNDA QAPKZher_(2: 25)VDNKFNK EMVDAGAEIWR LPNLNA KQM*AFIDSLGD DPSQ SANLLAEAKKLNDA QAPK

CD20 targeting antibodies include Rituximab, Ofatumumab (Arzerra®),Tositumumab (GSK), Ibritumomab (IDEC) (reference Ivanov 2008), 2F2(HuMax-CD20), 7D8, IgM2C6, IgG1 2C6, 11B8, B1, 2H7, LT20, 1F5 and AT80,as described in Teeling et al. (US 2004/0167319). Exemplary anti-CD20 VLand VH chains and whole antibodies are set forth below in Table C (Boldfonts indicate respective complimentary determining regions, CDRs, foreach chain, CDR1, CDR2 and CDR3):

TABLE C (SEQ ID NOs.: 48-69) V_(L) (Light chain):DIQMTQSPSSLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWTSNPPTFGQGTKVEIK V_(H) (heavy chain):EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVAAIYPGNGDTSYNQKFKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRSTYYGGDWYFDVWGQGTLVTVS S Rituzan®V_(L) and V_(H) chains. V_(L) (Light chain):QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK V_(H) (heavy chain):QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSIbritumomab (IDEC) V_(L) (Light chain):QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELK V_(H) (heavy chain):QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSTositumomab: >Mouse-Human chimeric Anti-CD20 (same V domains asibritumomab- they differ in constant regions only) V_(L) (Light chain):QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELK V_(H) (heavy chain):QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVS2C6 US7850962 B2 (Teeling et al) V_(H) (heavy chain):AVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGLGLEWVSGISWNSGSIGYADSVLGRFTISRDNALNSLYLQMNSLRAEDTALYYCTLDNQYGSGSTYGLGVWGQGTLVTVSSV_(L) (Light chain):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIL2F2 US2004/0167319A1 (Teeling et al) V_(H) (heavy chain):MFLGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS V_(L) (Light chain):MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK11B8 US2004/0167319A1 (Teeling, et al.) V_(H) (heavy chain):MELGLSWVFLVAILKGVQCEVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGLEWVSIIGTGGVTYYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAGSFYDGLYGMDVWGQGTTVTVSS V_(L) (Light chain):MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIK7D8 US2004/0167319A1 (Teeling et al) V_(H) (heavy chain):MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTISWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS V_(L) (Light chain):MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKWhole antibody drug sequences:Ibritumomab (IDEC) (reference Ivanov 2008)Mouse Anti-CD20 Heavy chain 1:QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRMouse Anti-CD20 Light chain 1:QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKRADAAPTVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN Rituximab Heavy chain chimeric:QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain chimeric:QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TositumomabMouse-Human chimeric Anti-CD20 Heavy Chain 1:QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mouse-Human chimeric Anti-CD20 Light Chain 1:QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

Non-limiting exemplary anti-luteinizing hormone releasing hormonereceptor (LHRH-R) Antibody Light (V_(L)) and Heavy (V_(H)) chainsequences are set for in Table D:

TABLE D (SEQ ID NOs.: 70-73)(A) Immunoglobulin light chains (κ) of GHR-106                    LEADERATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTACCTGTGGG M  D  S  Q  A  Q  V  L  I  L  L  L  L  W  V  S  G  T  C  G                         FR1GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACT D  I  V  M  S  Q  S  P  S  S  L  A  V  S  A  G  E  K  V  T                         CDR1ATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCT M  S  C  K  S  S  Q  S  L  L  N  S  R  T  R  K  N  Y  L  A                  PR2                        CDR2TGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGG W  Y  Q  Q  K  P  G  Q  S  P  K  L  L  I  Y  W  A  S  T  R                                FR3GAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACC E  S  G  V  P  D  R  F  T  G  S  G  S  G  T  D  F  T  L  T                                             CDR3ATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTT I  S  S  V  Q  A  E  D  L  A  V  Y  Y  C  K  Q  S  Y  N  L               FR4 TACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Y  T  F  G  G  G  T  K  L  E  I  K(B) Immunoglobulin heavy chains of GHR-106                                FR1CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCACCCTCACAGAGCCTGTCCATC Q  V  Q  L  K  E  S  G  P  G  L  V  A  P  S  Q  S  L  S  I                              CDR1ACATGCACTGTCTCTGGGTTCTCATTATCCAGATATAGTGTACACTGGGTTCGCCAGCCT T  C  T  V  S  G  E  S  L  S  R  Y  S  V  H  W  V  R  Q  P      FR2                             CDR2CCAGGAAAGGGCCTGGAGTGGCTGGGAATGATATGGGGTGGTGGAAGCACAGACTATAAT P  G  K  G  L  E  W  L  G  M  I  W  G  G  G  S  T  D  Y  N                                    FR3TCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTA S  A  L  K  S  R  L  S  I  S  K  D  N  S  K  S  Q  V  F  LAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGGCAATGAT K  M  N  S  L  Q  T  D  D  T  A  M  Y  Y  C  A  R  G  N  D      CDR3                FR4GGTTACTACTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA G  Y  Y  S  F  A  Y  W  G  Q  G  T  L  V  T  V  S  S

The invention includes modification or variations, such as amino acidmodifications (e.g., conservative or non-conservative substitutions,additions or deletions) of a conjugate, in an antibody, Heavy (H) chain,Light (L) chain, or fragment thereof portion, a lytic domain, or anyother domain of a antibody Heavy (H) chain, an antibody Light (L) chain,a fragment thereof, or a lytic domain can incorporate any number ofmodifications or variations, as long as such modifications or variationsdo not destroy target binding and/or lytic activity. Thus, for example,a modified antibody, Heavy (H) chain, Light (L) chain, or fragmentthereof, that binds to a target, such as a receptor (e.g., Her2/neu orCD20) can retain at least partial target (receptor) binding, or amodified lytic domain can retain at least partial lytic activity, suchas cell killing or apoptosis.

A “conservative substitution” is a replacement of one amino acid by abiologically, chemically or biological activity, e.g., lytic activity.Structurally similar means that the amino acids have side chains withsimilar length, such as alanine, glycine and serine, or having similarsize, or the structure of a first, second or additional domain ismaintained, such as an amphipathic alpha helix. Chemical similaritymeans that the residues have the same charge or are both hydrophilic andhydrophobic. Particular examples include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, serine for threonine, etc. Routine assayscan be used to determine whether a conjugate variant has activity, e.g.,target binding activity or lytic activity.

Modifications and variations therefore include of the various sequencesset forth herein, such as of antibodies, Heavy (H) chains and Light (L)chains of an antibody, and fragments thereof, as well as lytic domains(or additional domains, if present). Non-limiting modifications of lyticdomains are of KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA andKFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7), or a sequence thatincludes or consists of such lytic domains.

In particular embodiments, a subsequence of an antibody, Heavy (H)chain, Light (L) chain, fragments thereof, or a lytic domain has atleast 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35,35 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100,or more amino acid residues identical to the reference sequence. Inadditional particular embodiments, a substitution or deletion of one ormore amino acids (e.g., 1-3, 3-5, 5-10, 10-20, 20-30, or more) residuesof an antibody, Heavy (H) chain, Light (L) chain, fragment thereof,and/or lytic domain can have a sequence with 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or more identity to a reference sequence(e.g., antibody, Heavy (H) chain, Light (L) chain, fragment thereof, orlytic domain, such as KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,KFAKFAKKFAKFAKKFAKFA or KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7)).

In a particular embodiment, a conjugate includes an antibody, Heavy (H)chain, Light (L) chain, or fragment thereof, that binds to a target(e.g., receptor, such as Her2/neu or CD20) and a second lytic domainthat includes or consists of an L- or D-amino acid sequence set forthas: KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA orKFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7) having one or more of theK residues substituted with an F or L residue, one or more of the Fresidues substituted with a K, A or L residue, or one or more of the Aresidues substituted with a K, F or L residue. In another particularembodiment, a conjugate includes an antibody, Heavy (H) chain, Light (L)chain, or fragment thereof, that binds to a target (e.g., receptor, suchas Her2/neu or CD20) and a second domain consisting of an L- or D-aminoacid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ ID NOs.:1-7)having one or more of the K residues substituted with an F or L residue,one or more of the F residues substituted with a K, A or L residue, orone or more of the A residues substituted with a K, F or L residue(e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or28 residues in length).

The term “identity” and “homology” and grammatical variations thereofmean that two or more referenced entities are the same. Thus, where twoamino acid sequences are identical, they have the same amino acidsequence. “Areas, regions or domains of identity” mean that a portion oftwo or more referenced entities are the same. Thus, where two amino acidsequences are identical or homologous over one or more sequence regions,they share identity in these regions. The term “complementary,” whenused in reference to a nucleic acid sequence means the referencedregions are 100% complementary, i.e., exhibit 100% base pairing with nomismatches.

Due to variation in the amount of sequence conservation betweenstructurally and functionally related proteins, the amount of sequenceidentity required to retain a function or activity (e.g., target bindingor lytic) depends upon the protein, the region and the function oractivity of that region. For example, for a lytic peptide sequencemultiple PNNPNNP (SEQ ID NO.:153) sequence repeat patterns or motifs canbe present, but one or more interrupted or non-interrupted PNNPNNP (SEQID NO.:153) sequence repeat patterns or motifs need not be present.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm known in the art. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region. Forexample, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschulet al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI)has exemplary search parameters as follows: Mismatch −2; gap open 5; gapextension 2. For polypeptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

Conjugate amino acid residues can be joined by a covalent or anon-covalent bond. Non-limiting examples of covalent bonds are amidebonds, non-natural and non-amide chemical bonds, which include, forexample, glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups alternative to amidebonds include, for example, ketomethylene (e.g., —C(═O)—CH₂— for—C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether(CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide,thioamide, or ester (see, e.g., Spatola (1983) in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,“Peptide and Backbone Modifications,” Marcel Decker, N.Y.).

Lytic domains (and additional domains, when present) may be positionedanywhere on the antibody, antibody fragment, Heavy (H) chain, Light (L)chain, or fragment of a Heavy (H) chain or a Light (L) chain of anantibody, provided that the lytic domain (or any additional domain) doesnot destroy binding to a target. Non-limiting positions include a lyticdomain positioned at the amino-terminus, or at the carboxyl-terminus, ofan antibody Heavy (H) chain or a Light (L) chain. Where additionaldomains are present (e.g., third, fourth, fifth, sixth, seventh, etc.domains), the additional domain(s) can also be positioned anywhere.

First portion antibodies, Heavy (H) chains, Light (L) chains, andfragments thereof, and second (lytic) domains (and additional domains,when present), can be fused or joined to each other by a covalent or anon-covalent bond. First portion antibodies, Heavy (H) chains, Light (L)chains, and fragments thereof, and second (lytic) domains (andadditional domains, when present), can be immediately adjacent to eachother or separated by an intervening region, such as a hinge, spacer orlinker positioned between the two domains.

Examples of linkers or spacers include a non-peptide linker or spacer,such as a continuous carbon atom (C) chain (e.g., CCCCC). Multi-carbonchains include carboxylic acids (e.g., dicarboxylic acids) such asglutaric acid, succinic acid and adipic acid. A particular non-limitingexample is a 6 carbon linker such as α-amino-caproic acid.

Additional examples of linkers or spacers include one or more amino acidresidues, such as a peptide spacer or linker positioned between theantibody, Heavy (H) chain, Light (L) chainy, or fragment thereof, andthe second (lytic) domains (or additional domains, when present).Peptide spacer or linker sequences can be any length, but typicallyrange from about 1-10, 10-20, 20-30, 30-40, or 40-50 amino acidresidues. In particular embodiments, a peptide spacer or linkerpositioned between two (or more) domains is from 1 to 5, 1 to 10, 1 to20, 1 to 25 L- or D-amino acid residues, or 1 to 4, 1 to 6 or 1 to 8 L-or D-amino acid residues. Particular amino acid residues that areincluded in sequences positioned between the first and second domainsinclude one or more of or A, S or G amino acid residues. Specificnon-limiting examples of peptides positioned between the first andsecond domains include a sequence within or set forth as: GSGGS(SEQ IDNO.:9), ASAAS(SEQ ID NO.:8) or multiples of the particular linkersequence (GSGGS(SEQ ID NO.:9))n or (ASAAS(SEQ ID NO.:8))n, where n=1-5,5-10, 10-20, etc. Derivatives of amino acids and peptides can bepositioned between the two (or more) domains. A specific non-limitingexample of an amino acid derivative is a lysine derivative.

Conjugates with or without a spacer or linker, or a third, fourth,fifth, sixth, seventh, etc. domain can be entirely composed of naturalamino acids or synthetic, non-natural amino acids or amino acidanalogues, or can include derivatized forms. In various embodiments, aconjugate includes in a antibody, Heavy (H) chain, Light (L) chainy, orfragment thereof, and/or a second (lytic) domain (and additionaldomains, when present), one or more D-amino acids, mixtures of D-aminoacids and L-amino acids, or a sequence composed entirely of D-amino acidresidues.

Conjugates can contain any combination of non-natural structuralcomponents, which are typically from three structural groups: a) residuelinkage groups other than the natural amide bond (“peptide bond”)linkages; b) non-natural residues in place of naturally occurring aminoacid residues; or c) residues which induce secondary structural mimicry,i.e., induce or stabilize a secondary structure, e.g., an alpha helixconformation. Conjugates include cyclic structures such as an end-to-endamide bond between the amino and carboxy-terminus of the molecule orintra- or inter-molecular disulfide bond(s). Conjugates may be modifiedin vitro or in vivo, e.g., post-translationally modified to include, forexample, sugar or carbohydrate residues, phosphate groups, fatty acids,lipids, etc.

Specific examples of an addition include a third, fourth, fifth, sixthor seventh domain. Conjugates with two domains can therefore include oneor more additional domains (third, fourth, fifth, sixth, seventh, etc.)covalently linked thereto to impart a distinct or complementary functionor activity. Exemplary additional domains include domains facilitatingisolation, which include, for example, metal chelating peptides such aspolyhistidine tracts and histidine-tryptophan modules that allowpurification on immobilized metals; protein A binding domains that allowpurification on immobilized immunoglobulin; and domain utilized in theFLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). Optional inclusion of a cleavable sequence such as Factor Xa orenterokinase between a purification domain and the conjugate can be usedto facilitate purification. For example, an expression vector caninclude a conjugate-encoding nucleic acid sequence linked to sixhistidine residues followed by a thioredoxin and an enterokinasecleavage site. The histidine residues facilitate detection andpurification of the conjugate while the enterokinase cleavage siteprovides a means for purifying the construct from the remainder of theprotein (see e.g., Kroll, DNA Cell. Biol. 12:441 (1993)).

Conjugate activity can be affected by various factors and thereforeconjugates can be designed or optimized by taking into consideration oneor more of these factors. Such factors include, for example, length,which can affect toxicity to cells. Cell killing activity of alpha helixforming lytic peptide domains can also depend on the stability of thehelix. Linkers and spacers can affect membrane interaction of a lyticdomain and the helical structure of a lytic domain. The charge of lyticpeptide domains, which is determined in part by the particular aminoacid residues present in the domain, also affects cell killing potency.The positioning of antibody, Heavy (H) chain, Light (L) chain, andsecond (lytic) domains (and additional domains, when present), relativeto lytic domain (particular amino acid residue, or N- or C-terminus)also can affect cell killing activity of conjugates.

Conjugate in vivo half-life can be increased by constructing lyticpeptide domains with one or more non-naturally occurring amino acids orderivatives. For example, conjugates with D-amino acids (e.g., up to30%, 40%, 50%, 60%, or more of all residues are D-enantiomers) areresistant to serum proteolysis and therefore can be active for longertimes thereby increasing in vivo potency. Furthermore, constructinglytic peptide domains with one or more non-naturally occurring aminoacids or derivatives can reduce hemolytic activity. Such conjugates withD-enantiomers also have a greater tendency to be monomeric insolution—they do not significantly aggregate.

Peptides and peptidomimetics can be produced and isolated using methodsknown in the art. Peptides can be synthesized, whole or in part, usingchemical methods known in the art (see, e.g., Caruthers (1980). NucleicAcids Res. Symp. Ser. 215; Horn (1980); and Banga, A. K., TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems(1995) Technomic Publishing Co., Lancaster, Pa.). Peptide synthesis canbe performed using various solid-phase techniques (see, e.g., RobergeScience 269:202 (1995); Merrifield, Methods Enzymol. 289:3 (1997)) andautomated synthesis may be achieved, e.g., using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the manufacturer'sinstructions. Peptides and peptide mimetics can also be synthesizedusing combinatorial methodologies. Synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies known in the art (see, e.g., Organic SynthesesCollective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY).Modified peptides can be produced by chemical modification methods (see,for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, FreeRadic. Biol. Med 19:373 (1995); and Blommers, Biochemistry 33:7886(1994).

The invention further provides nucleic acids encoding the conjugates ofthe invention (and portions of antibodies, Heavy (H) chains, Light (L)chains, and fragments thereof, and second (lytic) domains, andadditional domains, when present), and vectors that include nucleic acidencoding conjugates. Nucleic acid, which can also be referred to hereinas a gene, polynucleotide, nucleotide sequence, primer, oligonucleotideor probe refers to natural or modified purine- and pyrimidine-containingpolymers of any length, either polyribonucleotides orpolydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides andca-anomeric forms thereof. The two or more purine- andpyrimidine-containing polymers are typically linked by a phosphoesterbond or analog thereof. The terms can be used interchangeably to referto all forms of nucleic acid, including deoxyribonucleic acid (DNA) andribonucleic acid (RNA). The nucleic acids can be single strand, double,or triplex, linear or circular. Nucleic acids include genomic DNA, cDNA,and antisense. RNA nucleic acid can be spliced or unspliced mRNA, rRNA,tRNA or antisense. Nucleic acids include naturally occurring, synthetic,as well as nucleotide analogues and derivatives.

As a result of the degeneracy of the genetic code, nucleic acids includesequences degenerate with respect to sequences encoding conjugates ofthe invention. Thus, degenerate nucleic acid sequences encodingconjugates are provided.

Nucleic acid can be produced using any of a variety of known standardcloning and chemical synthesis methods, and can be altered intentionallyby site-directed mutagenesis or other recombinant techniques known toone skilled in the art. Purity of polynucleotides can be determinedthrough sequencing, gel electrophoresis, UV spectrometry.

Nucleic acids may be inserted into a nucleic acid construct in whichexpression of the nucleic acid is influenced or regulated by an“expression control element,” referred to herein as an “expressioncassette.” The term “expression control element” refers to one or morenucleic acid sequence elements that regulate or influence expression ofa nucleic acid sequence to which it is operatively linked. An expressioncontrol element can include, as appropriate, promoters, enhancers,transcription terminators, gene silencers, a start codon (e.g., ATG) infront of a protein-encoding gene, etc.

An expression control element operatively linked to a nucleic acidsequence controls transcription and, as appropriate, translation of thenucleic acid sequence. The term “operatively linked” refers to ajuxtaposition wherein the referenced components are in a relationshippermitting them to function in their intended manner. Typicallyexpression control elements are juxtaposed at the 5′ or the 3′ ends ofthe genes but can also be intronic.

Expression control elements include elements that activate transcriptionconstitutively, that are inducible (i.e., require an external signal foractivation), or derepressible (i.e., require a signal to turntranscription off; when the signal is no longer present, transcriptionis activated or “derepressed”). Also included in the expressioncassettes of the invention are control elements sufficient to rendergene expression controllable for specific cell-types or tissues (i.e.,tissue-specific control elements). Typically, such elements are locatedupstream or downstream (i.e., 5′ and 3′) of the coding sequence.Promoters are generally positioned 5′ of the coding sequence. Promoters,produced by recombinant DNA or synthetic techniques, can be used toprovide for transcription of the polynucleotides of the invention. A“promoter” is meant a minimal sequence element sufficient to directtranscription.

Nucleic acids may be inserted into a plasmid for propagation into a hostcell and for subsequent genetic manipulation if desired. A plasmid is anucleic acid that can be stably propagated in a host cell; plasmids mayoptionally contain expression control elements in order to driveexpression of the nucleic acid. A vector is used herein synonymouslywith a plasmid and may also include an expression control element forexpression in a host cell. Plasmids and vectors generally contain atleast an origin of replication for propagation in a cell and a promoter.Plasmids and vectors are therefore useful for genetic manipulation ofconjugate encoding nucleic acids, producing conjugates or antisensenucleic acid, and expressing conjugates in host cells and organisms, forexample.

Bacterial system promoters include T7 and inducible promoters such as pLof bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) andtetracycline responsive promoters. Insect cell system promoters includeconstitutive or inducible promoters (e.g., ecdysone). Mammalian cellconstitutive promoters include SV40, RSV, bovine papilloma virus (BPV)and other virus promoters, or inducible promoters derived from thegenome of mammalian cells (e.g., metallothionein IIA promoter; heatshock promoter) or from mammalian viruses (e.g., the adenovirus latepromoter; the inducible mouse mammary tumor virus long terminal repeat).Alternatively, a retroviral genome can be genetically modified forintroducing and directing expression of a conjugate in appropriate hostcells.

Expression systems further include vectors designed for in vivo use.Particular non-limiting examples include adenoviral vectors (U.S. Pat.Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat. No.5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703),BPV vectors (U.S. Pat. No. 5,719,054) and CMV vectors (U.S. Pat. No.5,561,063).

Yeast vectors include constitutive and inducible promoters (see, e.g.,Ausubel et al., In: Current Protocols in Molecular Biology, Vol. 2, Ch.13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al.Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; BitterMethods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad.Press, N.Y.; and, Strathern et al., The Molecular Biology of the YeastSaccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). Aconstitutive yeast promoter such as ADH or LEU2 or an inducible promotersuch as GAL may be used (R. Rothstein In: DNA Cloning, A PracticalApproach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C.,1986). Vectors that facilitate integration of foreign nucleic acidsequences into a yeast chromosome, via homologous recombination forexample, are known in the art. Yeast artificial chromosomes (YAC) aretypically used when the inserted polynucleotides are too large for moreconventional vectors (e.g., greater than about 12 Kb).

Expression vectors also can contain a selectable marker conferringresistance to a selective pressure or identifiable marker (e.g.,beta-galactosidase), thereby allowing cells having the vector to beselected for, grown and expanded. Alternatively, a selectable marker canbe on a second vector that is cotransfected into a host cell with afirst vector containing a nucleic acid encoding a conjugate.

Selection systems include but are not limited to herpes simplex virusthymidine kinase gene (Wigler et al., Cell 11:223 (1977)),hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al.,Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes whichcan be employed in tk-, hgprt- or aprt-cells, respectively.Additionally, antimetabolite resistance can be used as the basis ofselection for dhfr, which confers resistance to methotrexate (O'Hare etal., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, whichconfers resistance to mycophenolic acid (Mulligan et al., Proc. Natl.Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistanceto aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1(1981)); puromycin; and hygromycin gene, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984)). Additional selectablegenes include trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988));and ODC (ornithine decarboxylase), which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue (1987) In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory).

Host cells that express conjugates, and host cells transformed withnucleic acids encoding conjugates (e.g., antibodies, Heavy (H) chains,Light (L) chains, and fragments thereof, and second (lytic) domains, andadditional domains, when present), and vectors including a nucleic acidthat encodes the conjugates are also provided. In one embodiment, a hostcell is a prokaryotic cell. In another embodiment, a host cell is aeukaryotic cell. In various aspects, the eukaryotic cell is a yeast ormammalian (e.g., human, primate, etc.) cell.

As used herein, a “host cell” is a cell into which a nucleic acid isintroduced that can be propagated, transcribed, or encoded conjugateexpressed. The term also includes any progeny or subclones of the hostcell. Host cells include cells that express conjugates.

Host cells include but are not limited to microorganisms such asbacteria and yeast; and plant, insect and mammalian cells. For example,bacteria transformed with recombinant bacteriophage nucleic acid,plasmid nucleic acid or cosmid nucleic acid expression vectors; yeasttransformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); and animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered fortransient or stable propagation or expression.

Antibody and polypeptide conjugates, nucleic acids encoding conjugates,and vectors and host cells expressing conjugates or transformed withnucleic acids encoding conjugates include isolated and purified forms.The term “isolated,” when used as a modifier of an invention conjugateor composition, means that the composition is made by the hand of man oris separated, substantially completely or at least in part, from thenaturally occurring in vivo environment. Generally, an isolatedcomposition is substantially free of one or more materials with which itnormally associates with in nature, for example, one or more protein,nucleic acid, lipid, carbohydrate, cell membrane. The term “isolated”does not exclude alternative physical forms of the composition, such asmultimers/oligomers, variants, modifications or derivatized forms, orforms expressed in host cells produced by the hand of man. The term“isolated” also does not exclude forms (e.g., pharmaceuticalformulations and combination compositions) in which there arecombinations therein, any one of which is produced by the hand of man.

An “isolated” composition can also be “purified” when free of some, asubstantial number of, most or all of the materials with which ittypically associates with in nature. Thus, an isolated conjugate thatalso is substantially pure does not include polypeptides orpolynucleotides present among millions of other sequences, such asproteins of a protein library or nucleic acids in a genomic or cDNAlibrary, for example. A “purified” composition can be combined with oneor more other molecules.

In accordance with the invention, there are provided mixtures ofconjugates and combination compositions. In one embodiment, a mixtureincludes one or more conjugates and a pharmaceutically acceptablecarrier or excipient. In another embodiment, a mixture includes one ormore conjugates and an anti-cell proliferative, anti-tumor, anti-cancer,or anti-neoplastic treatment or agent. In a further embodiment, amixture includes one or more conjugates and an immune enhancing agent.Combinations, such as one or more conjugates in a pharmaceuticallyacceptable carrier or excipient, with one or more of an anti-cellproliferative, anti-tumor, anti-cancer, or anti-neoplastic treatment oragent, and an immune enhancing treatment or agent, are also provided.

Conjugates of the invention, such as antibodies, Heavy (H) chains, Light(L) chains, and fragments thereof that bind to a target (e.g., areceptor), and second (lytic) domains, can be used to target cells forlysis, cell death or apoptosis. Such cells can be selectively targeted.For example a cell that expresses receptor such as Her2/neu or CD20 canbe targeted by a conjugate and thereby be preferentially killed comparedto cells that express little if any receptor.

In accordance with the invention, there are provided methods and uses ofreducing or inhibiting proliferation of a cell that expresses a target(e.g., a receptor) and methods of reducing or inhibiting cellproliferation. In one embodiment, a method or use includes contacting atarget expressing cell with a conjugate in an amount sufficient toreduce or inhibit proliferation of the cell. In another embodiment, amethod includes contacting a target expressing cell with a conjugate inan amount sufficient to reduce or inhibit cell proliferation.

Also provided are methods and uses of reducing or inhibitingproliferation of a hyperproliferative cell that expresses a target(e.g., a receptor), and methods and uses of reducing or inhibitingproliferation of hyperproliferating cells that express a target (e.g., areceptor). In one embodiment, a method or use includes contacting ahyperproliferative target expressing cell or hyperproliferating targetexpressing cells with a conjugate in an amount sufficient to reduce orinhibit proliferation.

Further provided are methods and uses of reducing or inhibitingproliferation of a non-metastatic or metastatic neoplastic, cancer,tumor and malignant cells that express a target (e.g., a receptor). Inone embodiment, a method or use includes contacting a neoplastic,cancer, tumor or malignant target expressing cell with a conjugate in anamount sufficient to reduce or inhibit proliferation of the targetexpressing cell.

Still further provided are methods and uses of reducing or inhibitingproliferation of a dormant or non-dividing non-metastatic or metastaticneoplastic, cancer, tumor and malignant cells that express a target(e.g., a receptor). In one embodiment, a method or use includescontacting a dormant or non-dividing neoplastic, cancer, tumor ormalignant target expressing cell with a conjugate in an amountsufficient to reduce or inhibit proliferation of the dormant ornon-dividing cell.

Additionally provided are methods and uses of selectively reducing orinhibiting proliferation of a cell (e.g., a hyperproliferating cell)that expresses a target (e.g., a receptor). In one embodiment, a methodor use includes contacting the target expressing cell with a conjugatein an amount sufficient to reduce or inhibit proliferation of the cell(e.g., hyperproliferating cell), wherein the conjugate binds to a target(e.g., a receptor) expressed by the cell.

Yet additionally provided are methods and uses of selectively reducingor inhibiting proliferation of a neoplastic, tumor, cancer or malignantcell that expresses that expresses a target (e.g., a receptor). In oneembodiment, a method or a use includes contacting the cell with aconjugate in an amount sufficient to reduce or inhibit proliferation ofthe neoplastic, tumor, cancer or malignant cell, wherein the conjugatebinds to the target (e.g., a receptor) expressed by the cell.

The term “contacting” means direct or indirect binding or interactionbetween two or more entities (e.g., between a conjugate and a targetand/or cell). Contacting as used herein includes in solution, in solidphase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo canbe referred to as administering, or administration or delivery.

Cells to target for reducing or inhibiting proliferation,non-selectively or selectively, include cells that express a target(e.g., a receptor). Non-limiting exemplary cells include breast,ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder,glial, dermal (e.g., melanocytes), hematologic and endometrial cells.

Conjugates and methods and uses of the invention are also applicable totreating undesirable or aberrant cell proliferation andhyperproliferative disorders, which include cells expressing a target(e.g., a receptor). Thus, in accordance with the invention, methods anduses of treating undesirable or aberrant cell proliferation andhyperproliferative disorders are provided. In one embodiment, a methodor a use includes administering to a subject (in need of treatment) anamount of a conjugate sufficient to treat the undesirable or aberrantcell proliferation or the hyperproliferative disorder.

The term “hyperproliferative disorder” refers to any undesirable oraberrant cell survival (e.g., failure to undergo programmed cell deathor apoptosis), growth or proliferation. Such disorders include benignhyperplasias, non-metastatic and metastatic neoplasias, cancers, tumorsand malignancies. Undesirable or aberrant cell proliferation andhyperproliferative disorders can affect any cell, tissue, organ in asubject. Undesirable or aberrant cell proliferation andhyperproliferative disorders can be present in a subject, locally,regionally or systemically. A hyperproliferative disorder can arise froma multitude of tissues and organs, including but not limited to breast,lung (e.g., small cell or non-small cell), thyroid, head and neck,brain, nasopharynx, throat, nose or sinuses, lymphoid, adrenal gland,pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus,stomach, duodenum, ileum, jejunum (small intestine), colon, rectum),genito-urinary tract (uterus, ovary, vagina, cervix, endometrium,fallopian tube, bladder, testicle, penis, prostate), kidney, pancreas,liver, bone, bone marrow, lymph, blood (hematologic), brain (glial),muscle, skin, dermal (e.g., melanocytes), and stem cells, which may ormay not metastasize to other secondary sites, regions or locations.

Conjugates and methods and uses of the invention are also applicable tometastatic or non-metastatic tumor, cancer, malignancy or neoplasia ofany cell, organ or tissue origin. Such disorders can affect virtuallyany cell or tissue type, e.g., carcinoma, sarcoma, melanoma, neural, andreticuloendothelial or hematopoietic neoplastic disorders (e.g.,myeloma, lymphoma or leukemia).

As used herein, the terms “neoplasia” and “tumor” refer to a cell orpopulation of cells whose growth, proliferation or survival is greaterthan growth, proliferation or survival of a normal counterpart cell,e.g. a cell proliferative or differentiative disorder. A tumor is aneoplasia that has formed a distinct mass or growth. A “cancer” or“malignancy” refers to a neoplasia or tumor that can invade adjacentspaces, tissues or organs. A “metastasis” refers to a neoplasia, tumor,cancer or malignancy that has disseminated or spread from its primarysite to one or more secondary sites, locations or regions within thesubject, in which the sites, locations or regions are distinct from theprimary tumor or cancer. All or a portion of such cells can express atarget (e.g., a receptor) can therefore be targeted with conjugates inaccordance with the invention.

Neoplastic, tumor, cancer and malignant cells (metastatic ornon-metastatic) include dormant or residual neoplastic, tumor, cancerand malignant cells, all or a portion of which express a target (e.g., areceptor). Such cells typically consist of remnant tumor cells that arenot dividing (G0-G1 arrest). These cells can persist in a primary siteor as disseminated neoplastic, tumor, cancer or malignant cells as aminimal residual disease. These dormant neoplastic, tumor, cancer ormalignant cells remain asymptomatic, but can develop severe symptoms anddeath once these dormant cells proliferate. Invention methods can beused to reduce or inhibit proliferation of dormant neoplastic, tumor,cancer or malignant cells, which can in turn inhibit or reduce tumor orcancer relapse, or tumor or cancer metastasis or progression.

In accordance with the invention, methods of treating a subject having ametastatic or non-metastatic tumor, cancer, malignancy or neoplasia areprovided. In one embodiment, a method includes administering to asubject (in need of treatment) an amount of a conjugate of sufficient totreat (e.g., reduce or inhibit proliferation) the metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia.

The metastatic or non-metastatic tumor, cancer, malignancy or neoplasiamay be in any stage, e.g., early or advanced, such as a stage I, II,III, IV or V tumor. The metastatic or non-metastatic tumor, cancer,malignancy or neoplasia may have been subject to a prior treatment or bestabilized (non-progressing) or in remission.

In terms of metastasis, invention methods can be used to reduce orinhibit metastasis of a primary tumor or cancer to other sites, or theformation or establishment of metastatic tumors or cancers at othersites distal from the primary tumor or cancer thereby inhibiting orreducing tumor or cancer relapse or tumor or cancer progression. Thus,methods of the invention include, among other things, 1) reducing orinhibiting growth, proliferation, mobility or invasiveness of tumor orcancer cells that potentially or do develop metastases (e.g.,disseminated tumor cells, DTC); 2) reducing or inhibiting formation orestablishment of metastases arising from a primary tumor or cancer toone or more other sites, locations or regions distinct from the primarytumor or cancer; 3) reducing or inhibiting growth or proliferation of ametastasis at one or more other sites, locations or regions distinctfrom the primary tumor or cancer after a metastasis has formed or hasbeen established; and 4) reducing or inhibiting formation orestablishment of additional metastasis after the metastasis has beenformed or established.

Cells of a metastatic or non-metastatic tumor, cancer, malignancy orneoplasia (all or a portion of which express a target (e.g., areceptor)) may be aggregated in a “solid” cell mass or be dispersed ordiffused. A “solid” tumor refers to cancer, neoplasia or metastasis thattypically aggregates together and forms a mass. Specific non-limitingexamples include breast, ovarian, uterine, cervical, stomach, lung,gastric, colon, bladder, glial, dermal (e.g., melanocytes) andendometrial tumors/cancers.

Carcinomas, which refer to malignancies of epithelial or endocrinetissue, include respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas. Exemplary carcinomas include those forming from theuterus, cervix, lung, prostate, breast, head and neck, colon, pancreas,testes, adrenal, kidney, esophagus, stomach, liver and ovary. The termalso includes carcinosarcomas, e.g., which include malignant tumorscomposed of carcinomatous and sarcomatous tissues. Adenocarcinomaincludes a carcinoma of a glandular tissue, or in which the tumor formsa gland like structure.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.

Neural neoplasias include glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma and oligodendrocytoma.

A “liquid tumor,” which refers to neoplasia that is dispersed or isdiffuse in nature, as they do not typically form a solid mass.Particular examples include neoplasia of the reticuloendothelial orhematopoietic system, such as lymphomas, myelomas and leukemias.Non-limiting examples of leukemias include acute and chroniclymphoblastic, myeloblastic and multiple myeloma. Typically, suchdiseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Specificmyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML). Lymphoid malignancies include, but are notlimited to, acute lymphoblastic leukemia (ALL), which includes B-lineageALL (B-ALL) and T-lineage ALL (T-ALL), chronic lymphocytic leukemia(CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstroem's macroglobulinemia (WM). Specific malignant lymphomasinclude, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

As disclosed herein, undesirable or aberrant cell proliferation orhyperproliferative disorders can occur in uterus, breast, vagina,cervix, endometrium and fallopian tube. Thus, in accordance with theinvention, there are provided methods and uses of treating uterus,breast, vagina, cervix, endometrium and fallopian tubehyperproliferative disorders. In one embodiment, a method or useincludes administering to a subject an amount of a conjugate sufficientto treat a uterus, breast, vagina, cervix, endometrium or fallopian tubehyperproliferative disorder.

Any composition, treatment, protocol, therapy or regimen having ananti-cell proliferative activity or effect can be combined with aconjugate or used in combination in a method or use of the invention.Conjugates and methods and uses of the invention therefore includeanti-proliferative, anti-tumor, anti-cancer, anti-neoplastic andanti-metastatic treatments, protocols and therapies, which include anyother composition, treatment, protocol or therapeutic regimen thatinhibits, decreases, retards, slows, reduces or prevents ahyperproliferative disorder, such as tumor, cancer, malignant orneoplastic growth, progression, metastasis, proliferation or survival,or worsening in vitro or in vivo. Particular non-limiting examples of ananti-proliferative (e.g., tumor) therapy include chemotherapy,immunotherapy, radiotherapy (ionizing or chemical), local thermal(hyperthermia) therapy, surgical resection and vaccination. A conjugatecan be administered prior to, substantially contemporaneously with orfollowing administration or use of the anti-cell proliferative,anti-neoplastic, anti-tumor, anti-cancer, anti-metastatic orimmune-enhancing treatment or therapy. A conjugate can be administeredor used as a combination composition with the anti-cell proliferative,anti-neoplastic, anti-tumor, anti-cancer, anti-metastatic orimmune-enhancing treatment or therapy, metastatic or non-metastatictumor, cancer, malignancy or neoplasia.

Anti-proliferative, anti-neoplastic, anti-tumor, anti-cancer andanti-metastatic compositions, therapies, protocols or treatments includethose that prevent, disrupt, interrupt, inhibit or delay cell cycleprogression or cell proliferation; stimulate or enhance apoptosis orcell death, inhibit nucleic acid or protein synthesis or metabolism,inhibit cell division, or decrease, reduce or inhibit cell survival, orproduction or utilization of a necessary cell survival factor, growthfactor or signaling pathway (extracellular or intracellular).Non-limiting examples of chemical agent classes having anti-cellproliferative, anti-neoplastic, anti-tumor, anti-cancer andanti-metastatic activities include alkylating agents, anti-metabolites,plant extracts, plant alkaloids, nitrosoureas, hormones, nucleoside andnucleotide analogues. Specific examples of drugs having anti-cellproliferative, anti-neoplastic, anti-tumor, anti-cancer andanti-metastatic activities include cyclophosphamide, azathioprine,cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine,busulphan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil,cytosine arabinoside, AZT, 5-azacytidine (5-AZC) and 5-azacytidinerelated compounds such as decitabine (5-aza-2′deoxycytidine),cytarabine, 1-beta-D-arabinofuranosyl-5-azacytosine anddihydro-5-azacytidine, bleomycin, actinomycin D, mithramycin, mitomycinC, carmustine, lomustine, semustine, streptozotocin, hydroxyurea,cisplatin, mitotane, procarbazine, dacarbazine, a taxane (e.g., taxol orpaclitaxel), vinblastine, vincristine, doxorubicin and dibromomannitoletc.

Additional agents that are applicable with conjugates and methods anduses are known to the skilled artisan and can be employed. For example,biologicals such as antibodies that are different from the antibodiesused for conjugation, cell growth factors, cell survival factors, celldifferentiative factors, cytokines and chemokines can be administered orused. Non-limiting examples of monoclonal antibodies include rituximab(Rituxan®), trastuzumab (Herceptin®), pertuzumab (Perjeta®)),bevacizumab (Avastin®), ranibizumab (Lucentis®), cetuximab (Erbitux®),alemtuzumab (Campath®), panitumumab (Vectibix®), ibritumomab tiuxetan(Zevalin®), tositumomab (Bexxar®), ipilimumab, zalutumumab, dalotuzumab,figitumumab, ramucirumab, galiximab, farletuzumab, ocrelizumab,ofatumumab (Arzerra®), tositumumab, ibritumomab, 2F2 (HuMax-CD20), 7D8,IgM2C6, IgG1 2C6, 11B8, B1, 2H7, LT20, 1F5 or AT80 daclizumab(Zenapax®), anti-LHRH receptor antibodies such as clone A9E4, F1G4,AT2G7, GNRH03, GNRHR2, etc. which can be used in combination with, interalia, a conjugate in accordance with the invention.

Other targeted drugs that are applicable for use with the conjugates arekinase inhibitors e.g., imatinib (Gleevec®), gefitinib (Iressa®),bortzomib (Velcade®), lapatinib (Tykerb®), sunitinib (Sutent®),sorafenib (Nevaxar®), nilotinib (Tasigna®) etc. Non-limiting examples ofcell growth factors, cell survival factors, cell differentiativefactors, cytokines and chemokines include IL-2, IL-1α, IL-1β, IL-3,IL-6, IL-7, granulocyte-macrophage-colony stimulating factor (GMCSF),IFN-γ, IL-12, TNF-α, TNFβ, MIP-1α, MIP-1β, RANTES, SDF-1, MCP-1, MCP-2,MCP-3, MCP-4, eotaxin, eotaxin-2, I-309/TCA3, ATAC, HCC-1, HCC-2, HCC-3,LARC/MIP-3α, PARC, TARC, CKβ, CKβ6, CKβ7, CKβ8, CKβ9, CKβ11, CKβ12, C10,IL-8, GROα, GROβ, ENA-78, GCP-2, PBP/CTAPIII□-TG/NAP-2, Mig, PBSF/SDF-1and lymphotactin.

Additional non-limiting examples include immune-enhancing treatments andtherapies, which include cell based therapies. In particular,immune-enhancing treatments and therapies include administeringlymphocytes, plasma cells, macrophages, dendritic cells, NK cells andB-cells.

Methods and uses of treating a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia, methods and uses of treating a subjectin need of treatment due to having or at risk of having a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia, and methods anduses of increasing effectiveness or improving an anti-proliferative,anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy, therapy areprovided. In respective embodiments, a method or use includesadministering to a subject with or at risk of a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia, an amount of aconjugate sufficient to treat the metastatic or non-metastatic tumor,cancer, malignancy or neoplasia; administering to the subject an amountof a conjugate sufficient to treat the subject; and administering to asubject that is undergoing or has undergone metastatic or non-metastatictumor, cancer, malignancy or neoplasia therapy, an amount of a conjugatesufficient to increase effectiveness of the anti-proliferative,anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy therapy.

Methods and uses of the invention may be practiced prior to (i.e.prophylaxis), concurrently with or after evidence of the presence ofundesirable or aberrant cell proliferation or a hyperproliferativedisorder, disease or condition begins (e.g., one or more symptoms).Administering or using a conjugate prior to, concurrently with orimmediately following development of a symptom of undesirable oraberrant cell proliferation or a hyperproliferative disorder maydecrease the occurrence, frequency, severity, progression, or durationof one or more symptoms of the undesirable or aberrant cellproliferation or a hyperproliferative disorder, disease or condition inthe subject. In addition, administering or using a conjugate prior to,concurrently with or immediately following development of one or moresymptoms of the undesirable or aberrant cell proliferation or ahyperproliferative disorder, disease or condition may inhibit, decreaseor prevent the spread or dissemination of hyperproliferating cells(e.g., metastasis) to other sites, regions, tissues or organs in asubject, or establishment of hyperproliferating cells (e.g., metastasis)at other sites, regions, tissues or organs in a subject.

Conjugates and the methods and uses of the invention, such as treatmentmethods and uses, can provide a detectable or measurable therapeuticbenefit or improvement to a subject. A therapeutic benefit orimprovement is any measurable or detectable, objective or subjective,transient, temporary, or longer-term benefit to the subject orimprovement in the condition, disorder or disease, an adverse symptom,consequence or underlying cause, of any degree, in a tissue, organ, cellor cell population of the subject.

Therapeutic benefits and improvements include, but are not limited to,reducing or decreasing occurrence, frequency, severity, progression, orduration of one or more symptoms or complications associated with adisorder, disease or condition, or an underlying cause or consequentialeffect of the disorder, disease or condition. Conjugates and methods anduses of the invention therefore include providing a therapeutic benefitor improvement to a subject.

In a method or use of the invention in which a therapeutic benefit orimprovement is a desired outcome, a conjugate can be administered in asufficient or effective amount to a subject in need thereof. An “amountsufficient” or “amount effective” refers to an amount that isanticipated to provide, in single or multiple doses, alone or incombination, with one or more other compositions (therapeutic agentssuch as a chemotherapeutic or immune stimulating drug), treatments,protocols, or therapeutic regimens agents, a detectable response of anyduration of time (long or short term), a desired outcome in or a benefitto a subject of any measurable or detectable degree or for any durationof time (e.g., for hours, days, months, years, or cured). The doses or“sufficient amount” or “effective amount” for treatment (e.g., toprovide a therapeutic benefit or improvement) typically are effective toameliorate a disorder, disease or condition, or one, multiple or alladverse symptoms, consequences or complications of the disorder, diseaseor condition, to a measurable extent, although reducing or inhibiting aprogression or worsening of the disorder, disease or condition or asymptom, is considered a satisfactory outcome.

The term “ameliorate” means a detectable objective or subjectiveimprovement in a subject's condition. A detectable improvement includesa subjective or objective reduction in the occurrence, frequency,severity, progression, or duration of a symptom caused by or associatedwith a disorder, disease or condition, an improvement in an underlyingcause or a consequence of the disorder, disease or condition, or areversal of the disorder, disease or condition.

Treatments or uses can therefore result in inhibiting, reducing orpreventing a disorder, disease or condition, or an associated symptom orconsequence, or underlying cause; inhibiting, reducing or preventing aprogression or worsening of a disorder, disease, condition, symptom orconsequence, or underlying cause; or further deterioration or occurrenceof one or more additional symptoms of the disorder, disease condition,or symptom. Thus, a successful treatment outcome leads to a “therapeuticeffect,” or “benefit” or inhibiting, reducing or preventing theoccurrence, frequency, severity, progression, or duration of one or moresymptoms or underlying causes or consequences of a condition, disorder,disease or symptom in the subject. Treatment methods and uses affectingone or more underlying causes of the condition, disorder, disease orsymptom are therefore considered to be beneficial. Stabilizing orinhibiting progression or worsening of a disorder or condition is also asuccessful treatment outcome.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the condition, disorder or disease.Thus, a satisfactory endpoint is achieved when there is a stabilizationor an incremental improvement in a subject's condition, or a partialreduction in the occurrence, frequency, severity, progression, orduration, or inhibition or reversal, of one or more associated adversesymptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the condition, disorder or disease), of one or more ofthe physiological, biochemical or cellular manifestations orcharacteristics of the disorder or disease, over a short or longduration of time (hours, days, weeks, months, etc.).

In particular embodiments, a method or use of treatment results inpartial or complete destruction of a metastatic or non-metastatic tumor,cancer, malignant or neoplastic cell mass, volume, size or numbers ofcells; results in stimulating, inducing or increasing metastatic ornon-metastatic tumor, cancer, malignant or neoplastic cell necrosis,lysis or apoptosis; results in reducing metastatic or non-metastatictumor, cancer, malignant or neoplastic volume, size, cell mass; resultsin inhibiting or preventing progression or an increase in metastatic ornon-metastatic tumor, cancer, malignant or neoplastic volume, mass, sizeor cell numbers; results in inhibiting or decreasing the spread ordissemination of hyperproliferating cells (e.g., metastasis) to other(secondary) sites, regions, tissues or organs in a subject, orestablishment of hyperproliferating cells (e.g., metastasis) at other(secondary) sites, regions, tissues or organs in a subject; or resultsin prolonging lifespan of the subject. In additional particularembodiments, a method or use of treatment results in reducing ordecreasing severity, duration or frequency of an adverse symptom orcomplication associated with or caused by the metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia.

An amount sufficient or an amount effective can but need not be providedin a single administration or dose and, can but need not be,administered alone or in combination with another composition (e.g.,chemotherapeutic or immune enhancing or stimulating agent), treatment,protocol or therapeutic regimen. For example, the amount may beproportionally increased as indicated by the need of the subject, statusof the disorder, disease or condition treated or the side effects oftreatment. In addition, an amount sufficient or an amount effective neednot be sufficient or effective if given in single or multiple doseswithout a second composition (e.g., chemotherapeutic or immunestimulating agent), treatment, protocol or therapeutic regimen, sinceadditional doses, amounts or duration above and beyond such doses, oradditional compositions (e.g., chemotherapeutic or immune stimulatingagents), treatments, protocols or therapeutic regimens may be includedin order to be considered effective or sufficient in a given subject.Amounts considered sufficient also include amounts that result in areduction of the use of another treatment, therapeutic regimen orprotocol.

An amount sufficient or an amount effective need not be effective ineach and every subject treated, prophylactically or therapeutically, nora majority of treated subjects in a given group or population. As istypical for treatment or therapeutic methods, some subjects will exhibitgreater or less response to a given treatment, therapeutic regimen orprotocol. An amount sufficient or an amount effective refers tosufficiency or effectiveness in a particular subject, not a group or thegeneral population. Such amounts will depend in part upon the conditiontreated, such as the type or stage of undesirable or aberrant cellproliferation or hyperproliferative disorder (e.g., a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia), the therapeuticeffect desired, as well as the individual subject (e.g., thebioavailability within the subject, gender, age, etc.).

Particular non-limiting examples of therapeutic benefit or improvementfor undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia) include a reduction in cell size, massor volume, inhibiting an increase in cell size, mass or volume, aslowing or inhibition of worsening or progression, stimulating cellnecrosis, lysis or apoptosis, reducing or inhibiting neoplastic or tumormalignancy or metastasis, reducing mortality, and prolonging lifespan ofa subject. Thus, inhibiting or delaying an increase in cell size, mass,volume or metastasis (stabilization) can increase lifespan (reducemortality) even if only for a few days, weeks or months, even thoughcomplete ablation of the metastatic or non-metastatic tumor, cancer,malignancy or neoplasia has not occurred. Adverse symptoms andcomplications associated with a hyperproliferative disorder (e.g., ametastatic or non-metastatic tumor, cancer, malignancy or neoplasia)that can be reduced or decreased include, for example, pain, nausea,discomfort, lack of appetite, lethargy and weakness. A reduction in theoccurrence, frequency, severity, progression, or duration of a symptomof undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia), such as an improvement in subjectivefeeling (e.g., increased energy, appetite, reduced nausea, improvedmobility or psychological well being, etc.), are therefore all examplesof therapeutic benefit or improvement.

For example, a sufficient or effective amount of a conjugate isconsidered as having a therapeutic effect if administration results inless chemotherapeutic drug, radiation or immunotherapy being requiredfor treatment of undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia).

The term “subject” refers to animals, typically mammalian animals, suchas humans, non human primates (apes, gibbons, chimpanzees, orangutans,macaques), domestic animals (dogs and cats), farm animals (horses, cows,goats, sheep, pigs) and experimental animal (mouse, rat, rabbit, guineapig). Subjects include animal disease models, for example, animal modelsof undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia) for analysis of conjugates in vivo.

Subjects appropriate for treatment include those having or at risk ofhaving a metastatic or non-metastatic tumor, cancer, malignant orneoplastic cell, those undergoing as well as those who are undergoing orhave undergone anti-proliferative (e.g., metastatic or non-metastatictumor, cancer, malignancy or neoplasia) therapy, including subjectswhere the tumor is in remission. “At risk” subjects typically have riskfactors associated with undesirable or aberrant cell proliferation,development of hyperplasia (e.g., a tumor).

Particular examples of at risk or candidate subjects include those withcells that express a target (e.g., a receptor) to which a conjugate canbind, particularly where cells targeted for necrosis, lysis, killing ordestruction express greater numbers or amounts of a target (e.g., areceptor) than non-target cells. Such cells can be selectively orpreferentially targeted for necrosis, lysis or killing.

At risk subjects also include those that are candidates for and thosethat have undergone surgical resection, chemotherapy, immunotherapy,ionizing or chemical radiotherapy, local or regional thermal(hyperthermia) therapy, or vaccination. The invention is thereforeapplicable to treating a subject who is at risk of a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia or a complicationassociated with a metastatic or non-metastatic tumor, cancer, malignancyor neoplasia, for example, due to metastatic or non-metastatic tumor,cancer, malignancy or neoplasia reappearance or regrowth following aperiod of stability or remission.

Risk factors include gender, lifestyle (diet, smoking), occupation(medical and clinical personnel, agricultural and livestock workers),environmental factors (carcinogen exposure), family history (autoimmunedisorders, diabetes, etc.), genetic predisposition, etc. For example,subjects at risk for developing melanoma include excess sun exposure(ultraviolet radiation), fair skin, high numbers of naevi (dysplasticnevus), patient phenotype, family history, or a history of a previousmelanoma. Subjects at risk for developing cancer can therefore beidentified by lifestyle, occupation, environmental factors, familyhistory, and genetic screens for tumor associated genes, gene deletionsor gene mutations. Subjects at risk for developing breast cancer lackBrcal, for example. Subjects at risk for developing colon cancer haveearly age or high frequency polyp formation, or deleted or mutated tumorsuppressor genes, such as adenomatous polyposis coli (APC), for example.

Subjects also include those precluded from other treatments. Forexample, certain subjects may not be good candidates for surgicalresection, chemotherapy, immunotherapy, ionizing or chemicalradiotherapy, local or regional thermal (hyperthermia) therapy, orvaccination. Thus, candidate subjects for treatment in accordance withthe invention include those that are not a candidate for surgicalresection, chemotherapy, immunotherapy, ionizing or chemicalradiotherapy, local or regional thermal (hyperthermia) therapy, orvaccination.

Conjugates may be formulated in a unit dose or unit dosage form. In aparticular embodiment, a conjugate is in an amount anticipated to beeffective to treat a subject having undesirable or aberrant cellproliferation or a hyperproliferative disorder. In an additionalparticular embodiment, a conjugate is in an amount anticipated to beeffective to treat a subject having a metastatic or non-metastatictumor, cancer, malignancy or neoplasia. Exemplary unit doses range fromabout 1-25, 25-250, 250-500, 500-1000, 1000-2500, 2500-5000,5000-25,000, 5000-50,000 μg; and from about 1-25, 25-250, 250-500,500-1000, 1000-2500 or 2500-5000, 5000-25,000, 5000-50,000 or50,000-100,000 mg.

Conjugates and methods and uses of the invention may be contacted orprovided in vitro, ex vivo or in vivo. Conjugates can be administered toprovide the intended effect as a single or multiple dosages, forexample, in an effective or sufficient amount. Exemplary doses rangefrom about 1-25, 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000,5000-25,000, 5000-50,000, or 50,000-100,000 μg/kg, on consecutive days,or alternating days or intermittently. Single or multiple doses can beadministered on consecutive days, alternating days or intermittently.

Conjugates can be administered and methods may be practiced viasystemic, regional or local administration, by any route. For example, aconjugate can be administered systemically, regionally or locally,intravenously, orally (e.g., ingestion or inhalation), intramuscularly,intraperitoneally, intradermally, subcutaneously, intracavity,intracranially, transdermally (topical), parenterally, e.g.transmucosally or rectally. Compositions and methods and uses of theinvention including pharmaceutical formulations can be administered viaa (micro)encapsulated delivery system or packaged into an implant foradministration.

The invention further provides conjugates and methods and uses in whichthe conjugates are included in pharmaceutical compositions. Apharmaceutical composition refers to “pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. As usedherein, the term “pharmaceutically acceptable” and “physiologicallyacceptable,” when referring to carriers, diluents or excipients includessolvents (aqueous or non-aqueous), detergents, solutions, emulsions,dispersion media, coatings, isotonic and absorption promoting ordelaying agents, compatible with pharmaceutical administration and withthe other components of the formulation. Such formulations can becontained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration or use. Compositions for parenteral,intradermal, or subcutaneous administration can include a sterilediluent, such as water, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents. Thepreparation may contain one or more preservatives to preventmicroorganism growth (e.g., antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose).

Pharmaceutical compositions for injection include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and polyethylene glycol), andsuitable mixtures thereof. Fluidity can be maintained, for example, bythe use of a coating such as lecithin, or by the use of surfactants.Antibacterial and antifungal agents include, for example, parabens,chlorobutanol, phenol, ascorbic acid and thimerosal. Including an agentthat delays absorption, for example, aluminum monostearate and gelatincan prolonged absorption of injectable compositions. Polysorbate 20 andpolysorbate 80 can be added into the formulation mixture, for example,up to 1%. Other non-limiting additives include histidine HCl,α,α-treahlose dehydrate.

Additional pharmaceutical formulations and delivery systems are known tothe skilled artisan and are applicable in the methods of the invention(see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., MackPublishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., MerckPublishing Group, Whitehouse, N.J.; Pharmaceutical Principles of SolidDosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993);and Poznansky, et al., Drug Delivery Systems, R. L. Juliano, ed.,Oxford, N.Y. (1980), pp. 253-315).

The invention provides kits including conjugates of the invention,combination compositions and pharmaceutical formulations thereof,packaged into suitable packaging material. A kit optionally includes alabel or packaging insert including a description of the components orinstructions for use in vitro, in vivo, or ex vivo, of the componentstherein. Exemplary instructions include instructions for reducing orinhibiting proliferation of a cell, reducing or inhibiting proliferationof undesirable or aberrant cells, such as a hyperproliferating cell,reducing or inhibiting proliferation of a metastatic or non-metastatictumor, cancer, malignant or neoplastic cell, treating a subject having ahyperproliferative disorder, treating a subject having a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia, or reducingfertility of an animal.

A kit can contain a collection of such components, e.g., two or moreconjugates alone, or in combination with another therapeutically usefulcomposition (e.g., an anti-proliferative or immune-enhancing drug).

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.).

Kits of the invention can include labels or inserts. Labels or insertsinclude “printed matter,” e.g., paper or cardboard, or separate oraffixed to a component, a kit or packing material (e.g., a box), orattached to an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, opticaldisk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or anelectrical storage media such as RAM and ROM or hybrids of these such asmagnetic/optical storage media, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer information, lot numbers, manufacturer location and date.

Labels or inserts can include information on a condition, disorder,disease or symptom for which a kit component may be used. Labels orinserts can include instructions for the clinician or for a subject forusing one or more of the kit components in a method, treatment protocolor therapeutic regimen. Instructions can include dosage amounts,frequency or duration, and instructions for practicing any of themethods, treatment protocols or therapeutic regimes set forth herein.Exemplary instructions include, instructions for treating an undesirableor aberrant cell proliferation, hyperproliferating cells and disorders(e.g., metastatic or non-metastatic tumor, cancer, malignancy orneoplasia). Kits of the invention therefore can additionally includelabels or instructions for practicing any of the methods and uses of theinvention described herein.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, such as warnings to the subject or clinician regardingsituations where it would not be appropriate to use a particularcomposition. Adverse side effects could also occur when the subject has,will be or is currently taking one or more other medications that may beincompatible with the composition, or the subject has, will be or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the composition and, therefore,instructions could include information regarding such incompatibilities.

Invention kits can additionally include other components. Each componentof the kit can be enclosed within an individual container and all of thevarious containers can be within a single package. Invention kits can bedesigned for cold storage. Invention kits can further be designed tocontain host cells expressing conjugates of the invention, or thatcontain nucleic acids encoding conjugates. The cells in the kit can bemaintained under appropriate storage conditions until the cells areready to be used. For example, a kit including one or more cells cancontain appropriate cell storage medium so that the cells can be thawedand grown.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a conjugate” or “a target (e.g., a receptor),” ora “lytic domain” includes a plurality of such conjugates, targets, orlytic domains, and so forth.

As used herein, numerical values are often presented in a range formatthroughout this document. The use of a range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention unless the context clearlyindicates otherwise. Accordingly, a range expressly includes allpossible subranges, all individual numerical values within that range,and all numerical values or numerical ranges including integers withinsuch ranges and fractions of the values or the integers within rangesunless the context clearly indicates otherwise. This constructionapplies regardless of the breadth of the range and in all contextsthroughout this patent document. Thus, for example, reference to a rangeof 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%,91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100%also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%,91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%,etc., and so forth.

In addition, reference to a range of 1-5,000 fold includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc.,as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5,fold, etc., and any numerical range within such a ranges, such as 1-2,5-10, 10-50, 50-100, 100-500, 100-1000, 500-1000, 1000-2000, 1000-5000,etc. In a further example, reference to a range of KD 10-⁵ M to about KD10-¹³ M includes any numerical value or range within or encompassingsuch values.

As also used herein a series of ranges are disclosed throughout thisdocument. The use of a series of ranges include combinations of theupper and lower ranges to provide another range. This constructionapplies regardless of the breadth of the range and in all contextsthroughout this patent document. Thus, for example, reference to aseries of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75,75-100, 100-150, and 150-171, includes ranges such as 5-20, 5-30, 5-40,5-50, 5-75, 5-100, 5-150, 5-171, and 10-30, 10-40, 10-50, 10-75, 10-100,10-150, 10-171, and 20-40, 20-50, 20-75, 20-100, 20-150, 20-171, and soforth.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

To determine, in in vitro studies, cytotoxicity of recombinantlyproduced antibody (as a antibody) scFv-C_(H)3 to Her-2 receptorconjugated to the lytic peptide, Phor18 (KFAKFAK KFAKFAK KFAK (SEQ IDNO.:4)) or (KLAKLAK)2KLAK (SEQ ID NO.:74). Various linkers (GS andNRVRRS (SEQ ID NO.:75)) and 1 or 2 molecules of lytic peptides perantibody molecule were studied.

Peptides studied were: Phor18-scFv-C_(H)3-Phor18 (2 molecules of Phor-18joined at N- and C-terminal ends of the antibody, scFv-C_(H)3-GS-Phor18(one molecule of Phor18 joined to the antibody at the C-terminus by GSlinker, scFv-C_(H)3-GS-(KLAKLAK)₂KLAK (SEQ ID NO.:74) (one molecule of(KLAKLAK)₂KLAK (SEQ ID NO.:74) linked to the antibody at the C-terminusby GS linker, scFv-C_(H)3-NRVRRS (SEQ ID NO.:75)-Phor18 (one molecule ofPhor18 to the antibody at the C-terminus by NRVRRS linker, andscFv-C_(H)3-NRVRRS-(KLAKLAK)₂KLAK (SEQ ID NO.:74) (one molecule of(KLAKLAK)₂KLAK (SEQ ID NO.:74) to the antibody at the C-terminus byNRVRRS linker). Cytotoxicity was compared to a naked antibody (antibodywithout a lytic peptide) in Her-2 receptor positive cells (SKBR-3 andSKOV-3, human breast and ovarian cancer cells, respectively) and Her-2receptor negative breast cancer cells (MDA-MB-231).

Example 2

This example describes various materials and methods used in the studiesdescribed herein.

Materials:

Recombinant DNA technique was used to synthesize anti-Her2/neu antibodyas a recombinant antibody in Escherichia coli. The scFv-C_(H)3 antibody(Olafsen T. et al Protein Engineering, Design & Selection 17, 315-323,2004) was conjugated via a peptide linker or without a linker asdescribed in Table 1 to either Phor18 or an amphipathic, alpha-helicallytic peptide, (KLAKLAK)₂KLAK (SEQ ID NO.:74) and analyzed forcytotoxicity in vitro. The plasmid was acquired through gene codonoptimization. The gene was synthesized with a N-His tag sequence and theplasmid was subcloned into an E. coli bacteria expression vector pUC57.After expression optimization and evaluation the His-tag product wasselected and 1 L of the bacteria expression product was purified in aone-step affinity purification. The sequences of the plasmid geneinsertion for each construct is described in Table 1.

TABLE 1Nucleotide sequence of the plasmid insertion for the production of eachrecombinant Her2/neu antibody and antibody conjugate.1. Her2/neu scFv-C_(H)3: (SEQ ID NO.: 76) 1CATATGCATC ACCACCACCA CCACGACGAC GACGACAAAG ATATTCAAAT GACCCAGTCC 61CCGAGCAGCC TGAGTGCCTC CGTTGGCGAC CGCGTGACCA TTACGTGCCG TGCGAGCCAG 121GATGTCAACA CCGCGGTGGC CTGGTATCAG CAAAAACCGG GCAAAGCGCC GAAACTGCTG 181ATCTATTCAG CCTCGTTTCT GTACAGCGGT GTTCCGTCTC GTTTCAGCGG CTCTCGCAGT 241GGTACCGATT TTACCCTGAC GATTAGCTCT CTGCAGCCGG AAGACTTTGC GACGTATTAC 301TGCCAGCAAC ATTACACCAC GCCGCCGACC TTCGGCCAGG GTACGAAAGT GGAAATCAAA 361GGTTCCACCT CAGGCGGTGG CAGTGGTGGC GGTTCCGGCG GTGGCGGTAG TTCCGAAGTT 421CAGCTGGTCG AAAGTGGCGG TGGCCTGGTT CAACCGGGTG GCTCACTGCG TCTGTCGTGT 481GCAGCAAGCG GTTTCAACAT CAAAGATACC TACATCCACT GGGTTCGTCA GGCGCCGGGC 541AAAGGTCTGG AATGGGTCGC CCGCATTTAC CCGACCAATG GCTATACGCG TTACGCAGAT 601AGCGTGAAAG GTCGCTTTAC CATCTCTGCG GACACCAGTA AAAACACGGC CTATCTGCAG 661ATGAATAGCC TGCGTGCGGA AGATACGGCC GTTTATTACT GCTCTCGCTG GGGTGGCGAT 721GGCTTCTATG CTATGGACTA CTGGGGCCAG GGTACCCTGG TGACGGTTTC ATCGGGTCAG 781CCGCGTGAAC CGCAAGTGTA TACCCTGCCG CCGTCACGCG ATGAACTGAC GAAAAACCAG 841GTGTCGCTGA CGTGTCTGGT TAAAGGCTTT TACCCGAGCG ACATCGCGGT TGAATGGGAA 901TCTAATGGTC AACCGGAAAA CAATTATAAA ACCACGCCGC CGGTCCTGGA TAGTGACGGC 961TCCTTTTTCC TGTACAGTAA ACTGACCGTG GATAAATCCC GTTGGCAGCA GGGTAACGTC 1021TTCTCGTGTA GCGTGATGCA TGAAGCCCTG CATAATCACT ATACCCAGAA ATCTCTGAGT 1081CTGTCCCCGG GCAAAGGTTC AACGTCGGGT GGCGGTTCCG GCGGTGGCTC AGGTGGCGGT 1141GGCAGCTCTG GCCAACCGCG CGAACCGCAG GTTTACACCC TGCCGCCGAG CCGTGACGAA 1201CTGACCAAAA ACCAAGTCAG CCTGACGTGC CTGGTGAAAG GCTTTTACCC GAGTGACATT 1261GCAGTTGAAT GGGAATCCAA TGGTCAGCCG GAAAATAACT ACAAAACGAC GCCGCCGGTT 1321CTGGATTCAG ACGGCTCGTT TTTCCTGTAC TCAAAACTGA CCGTCGATAA ATCGCGCTGG 1381CAACAGGGTA ACGTTTTCAG CTGCTCTGTC ATGCACGAAG CCCTGCACAA CCATTATACC 1441CAGAAAAGTC TGTCCCTGTC ACCGGGCAAA GAAGTGCAGC TGGTTGAATC TGGTGGCGGT 1501CTGGTGCAAC CGGGCGGTTC GCTGCGTCTG AGCTGTGCAG CTTCTGGCTT TAATATTAAA 1561GACACGTACA TCCACTGGGT GCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTTGCTCGT 1621ATCTATCCGA CGAACGGTTA TACGCGTTAC GCCGATAGCG TCAAAGGCCG TTTTACCATC 1681AGTGCAGACA CCTCCAAAAA CACGGCTTAT CTGCAGATGA ATAGTCTGCG TGCAGAAGAT 1741ACCGCTGTTT ATTACTGCAG CCGCTGGGGC GGTGATGGCT TCTATGCAAT GGATTATTGG 1801GGTCAAGGTA CCCTGGTCAC CGTGAGTTCC GGTTCGACCA GCGGCGGTGG CTCAGGTGGC 1861GGTTCGGGCG GTGGCGGTTC ATCGGACATT CAGATGACGC AAAGCCCGAG CTCTCTGTCT 1921GCGAGTGTTG GCGATCGTGT CACCATCACG TGTCGCGCCT CTCAGGACGT GAATACCGCA 1981GTTGCTTGGT ACCAACAAAA ACCGGGCAAA GCACCGAAAC TGCTGATTTA CTCCGCTTCA 2041TTCCTGTACA GCGGTGTGCC GTCTCGTTTT TCGGGCAGCC GCTCTGGTAC CGATTTCACC 2101CTGACGATTA GTTCCCTGCA ACCGGAAGAT TTCGCCACCT ACTACTGCCA GCAACACTAT 2161ACGACCCCGC CGACGTTTGG TCAGGGCACG AAAGTGGAAA TTAAATAATG AAAGCTT2. scFv-C_(H)3-GS-Phor18: (SEQ ID NO.: 77) 1CATATGCATC ACCACCACCA CCACGACGAC GACGACAAAG ATATTCAAAT GACCCAGTCC 61CCGAGCAGCC TGAGTGCCTC CGTTGGCGAC CGCGTGACCA TTACGTGCCG TGCGAGCCAG 121GATGTCAACA CCGCGGTGGC CTGGTATCAG CAAAAACCGG GCAAAGCGCC GAAACTGCTG 181ATCTATTCAG CCTCGTTTCT GTACAGCGGT GTTCCGTCTC GTTTCAGCGG CTCTCGCAGT 241GGTACCGATT TTACCCTGAC GATTAGCTCT CTGCAGCCGG AAGACTTTGC GACGTATTAC 301TGCCAGCAAC ATTACACCAC GCCGCCGACC TTCGGCCAGG GTACGAAAGT GGAAATCAAA 361GGTTCCACCT CAGGCGGTGG CAGTGGTGGC GGTTCCGGCG GTGGCGGTAG TTCCGAAGTT 421CAGCTGGTCG AAAGTGGCGG TGGCCTGGTT CAACCGGGTG GCTCACTGCG TCTGTCGTGT 481GCAGCAAGCG GTTTCAACAT CAAAGATACC TACATCCACT GGGTTCGTCA GGCGCCGGGC 541AAAGGTCTGG AATGGGTCGC CCGCATTTAC CCGACCAATG GCTATACGCG TTACGCAGAT 601AGCGTGAAAG GTCGCTTTAC CATCTCTGCG GACACCAGTA AAAACACGGC CTATCTGCAG 661ATGAATAGCC TGCGTGCGGA AGATACGGCC GTTTATTACT GCTCTCGCTG GGGTGGCGAT 721GGCTTCTATG CTATGGACTA CTGGGGCCAG GGTACCCTGG TGACGGTTTC ATCGGGTCAG 781CCGCGTGAAC CGCAAGTGTA TACCCTGCCG CCGTCACGCG ATGAACTGAC GAAAAACCAG 841GTGTCGCTGA CGTGTCTGGT TAAAGGCTTT TACCCGAGCG ACATCGCGGT TGAATGGGAA 901TCTAATGGTC AACCGGAAAA CAATTATAAA ACCACGCCGC CGGTCCTGGA TAGTGACGGC 961TCCTTTTTCC TGTACAGTAA ACTGACCGTG GATAAATCCC GTTGGCAGCA GGGTAACGTC 1021TTCTCGTGTA GCGTGATGCA TGAAGCCCTG CATAATCACT ATACCCAGAA ATCTCTGAGT 1081CTGTCCCCGG GCAAAGGTTC AACGTCGGGT GGCGGTTCCG GCGGTGGCTC AGGTGGCGGT 1141GGCAGCTCTG GCCAACCGCG CGAACCGCAG GTTTACACCC TGCCGCCGAG CCGTGACGAA 1201CTGACCAAAA ACCAAGTCAG CCTGACGTGC CTGGTGAAAG GCTTTTACCC GAGTGACATT 1261GCAGTTGAAT GGGAATCCAA TGGTCAGCCG GAAAATAACT ACAAAACGAC GCCGCCGGTT 1321CTGGATTCAG ACGGCTCGTT TTTCCTGTAC TCAAAACTGA CCGTCGATAA ATCGCGCTGG 1381CAACAGGGTA ACGTTTTCAG CTGCTCTGTC ATGCACGAAG CCCTGCACAA CCATTATACC 1441CAGAAAAGTC TGTCCCTGTC ACCGGGCAAA GAAGTGCAGC TGGTTGAATC TGGTGGCGGT 1501CTGGTGCAAC CGGGCGGTTC GCTGCGTCTG AGCTGTGCAG CTTCTGGCTT TAATATTAAA 1561GACACGTACA TCCACTGGGT GCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTTGCTCGT 1621ATCTATCCGA CGAACGGTTA TACGCGTTAC GCCGATAGCG TCAAAGGCCG TTTTACCATC 1681AGTGCAGACA CCTCCAAAAA CACGGCTTAT CTGCAGATGA ATAGTCTGCG TGCAGAAGAT 1741ACCGCTGTTT ATTACTGCAG CCGCTGGGGC GGTGATGGCT TCTATGCAAT GGATTATTGG 1801GGTCAAGGTA CCCTGGTCAC CGTGAGTTCC GGTTCGACCA GCGGCGGTGG CTCAGGTGGC 1861GGTTCGGGCG GTGGCGGTTC ATCGGACATT CAGATGACGC AAAGCCCGAG CTCTCTGTCT 1921GCGAGTGTTG GCGATCGTGT CACCATCACG TGTCGCGCCT CTCAGGACGT GAATACCGCA 1981GTTGCTTGGT ACCAACAAAA ACCGGGCAAA GCACCGAAAC TGCTGATTTA CTCCGCTTCA 2041TTCCTGTACA GCGGTGTGCC GTCTCGTTTT TCGGGCAGCC GCTCTGGTAC CGATTTCACC 2101CTGACGATTA GTTCCCTGCA ACCGGAAGAT TTCGCCACCT ACTACTGCCA GCAACACTAT 2161ACGACCCCGC CGACGTTTGG TCAGGGCACG AAAGTGGAAA TTAAAGGCAG CAAATTTGCG 2221AAATTCGCCA AAAAATTCGC AAAATTCGCG AAAAAATTCG CGAAATAATG AAAGCTT3. scFv-C_(H)3-GS-(KLAKLAK)₂KLAK: (SEQ ID NO.: 78) 1CATATGGAAA ATCTGTATTT CCAAGGTGAT ATTCAAATGA CCCAGTCCCC GAGCAGCCTG 61AGTGCCTCCG TTGGCGACCG CGTGACCATT ACGTGCCGTG CGAGCCAGGA TGTCAACACC 121GCGGTGGCCT GGTATCAGCA AAAACCGGGC AAAGCGCCGA AACTGCTGAT CTATTCAGCC 181TCGTTTCTGT ACAGCGGTGT TCCGTCTCGT TTCAGCGGCT CTCGCAGTGG TACCGATTTT 241ACCCTGACGA TTAGCTCTCT GCAGCCGGAA GACTTTGCGA CGTATTACTG CCAGCAACAT 301TACACCACGC CGCCGACCTT CGGCCAGGGT ACGAAAGTGG AAATCAAAGG TTCCACCTCA 361GGCGGTGGCA GTGGTGGCGG TTCCGGCGGT GGCGGTAGTT CCGAAGTTCA GCTGGTCGAA 421AGTGGCGGTG GCCTGGTTCA ACCGGGTGGC TCACTGCGTC TGTCGTGTGC AGCAAGCGGT 481TTCAACATCA AAGATACCTA CATCCACTGG GTTCGTCAGG CGCCGGGCAA AGGTCTGGAA 541TGGGTCGCCC GCATTTACCC GACCAATGGC TATACGCGTT ACGCAGATAG CGTGAAAGGT 601CGCTTTACCA TCTCTGCGGA CACCAGTAAA AACACGGCCT ATCTGCAGAT GAATAGCCTG 661CGTGCGGAAG ATACGGCCGT TTATTACTGC TCTCGCTGGG GTGGCGATGG CTTCTATGCT 721ATGGACTACT GGGGCCAGGG TACCCTGGTG ACGGTTTCAT CGGGTCAGCC GCGTGAACCG 781CAAGTGTATA CCCTGCCGCC GTCACGCGAT GAACTGACGA AAAACCAGGT GTCGCTGACG 841TGTCTGGTTA AAGGCTTTTA CCCGAGCGAC ATCGCGGTTG AATGGGAATC TAATGGTCAA 901CCGGAAAACA ATTATAAAAC CACGCCGCCG GTCCTGGATA GTGACGGCTC CTTTTTCCTG 961TACAGTAAAC TGACCGTGGA TAAATCCCGT TGGCAGCAGG GTAACGTCTT CTCGTGTAGC 1021GTGATGCATG AAGCCCTGCA TAATCACTAT ACCCAGAAAT CTCTGAGTCT GTCCCCGGGC 1081AAAGGTTCAA CGTCGGGTGG CGGTTCCGGC GGTGGCTCAG GTGGCGGTGG CAGCTCTGGC 1141CAACCGCGCG AACCGCAGGT TTACACCCTG CCGCCGAGCC GTGACGAACT GACCAAAAAC 1201CAAGTCAGCC TGACGTGCCT GGTGAAAGGC TTTTACCCGA GTGACATTGC AGTTGAATGG 1261GAATCCAATG GTCAGCCGGA AAATAACTAC AAAACGACGC CGCCGGTTCT GGATTCAGAC 1321GGCTCGTTTT TCCTGTACTC AAAACTGACC GTCGATAAAT CGCGCTGGCA ACAGGGTAAC 1381GTTTTCAGCT GCTCTGTCAT GCACGAAGCC CTGCACAACC ATTATACCCA GAAAAGTCTG 1441TCCCTGTCAC CGGGCAAAGA AGTGCAGCTG GTTGAATCTG GTGGCGGTCT GGTGCAACCG 1501GGCGGTTCGC TGCGTCTGAG CTGTGCAGCT TCTGGCTTTA ATATTAAAGA CACGTACATC 1561CACTGGGTGC GTCAGGCACC GGGTAAAGGC CTGGAATGGG TTGCTCGTAT CTATCCGACG 1621AACGGTTATA CGCGTTACGC CGATAGCGTC AAAGGCCGTT TTACCATCAG TGCAGACACC 1681TCCAAAAACA CGGCTTATCT GCAGATGAAT AGTCTGCGTG CAGAAGATAC CGCTGTTTAT 1741TACTGCAGCC GCTGGGGCGG TGATGGCTTC TATGCAATGG ATTATTGGGG TCAAGGTACC 1801CTGGTCACCG TGAGTTCCGG TTCGACCAGC GGCGGTGGCT CAGGTGGCGG TTCGGGCGGT 1861GGCGGTTCAT CGGACATTCA GATGACGCAA AGCCCGAGCT CTCTGTCTGC GAGTGTTGGC 1921GATCGTGTCA CCATCACGTG TCGCGCCTCT CAGGACGTGA ATACCGCAGT TGCTTGGTAC 1981CAACAAAAAC CGGGCAAAGC ACCGAAACTG CTGATTTACT CCGCTTCATT CCTGTACAGC 2041GGTGTGCCGT CTCGTTTTTC GGGCAGCCGC TCTGGTACCG ATTTCACCCT GACGATTAGT 2101TCCCTGCAAC CGGAAGATTT CGCCACCTAC TACTGCCAGC AACACTATAC GACCCCGCCG 2161ACGTTTGGTC AGGGCACGAA AGTGGAAATT AAAGGCAGCA AACTGGCGAA ACTGGCCAAA 2221AAACTGGCAA AACTGGCGAA AAAACTGGCG AAATAATGAA AGCTT4. scFv-C_(H)3-NRVRRS-Phor18: (SEQ ID NO.: 79) 1CATATGGAAA ATCTGTATTT CCAAGGTGAT ATTCAAATGA CCCAGTCCCC GAGCAGCCTG 61AGTGCCTCCG TTGGCGACCG CGTGACCATT ACGTGCCGTG CGAGCCAGGA TGTCAACACC 121GCGGTGGCCT GGTATCAGCA AAAACCGGGC AAAGCGCCGA AACTGCTGAT CTATTCAGCC 181TCGTTTCTGT ACAGCGGTGT TCCGTCTCGT TTCAGCGGCT CTCGCAGTGG TACCGATTTT 241ACCCTGACGA TTAGCTCTCT GCAGCCGGAA GACTTTGCGA CGTATTACTG CCAGCAACAT 301TACACCACGC CGCCGACCTT CGGCCAGGGT ACGAAAGTGG AAATCAAAGG TTCCACCTCA 361GGCGGTGGCA GTGGTGGCGG TTCCGGCGGT GGCGGTAGTT CCGAAGTTCA GCTGGTCGAA 421AGTGGCGGTG GCCTGGTTCA ACCGGGTGGC TCACTGCGTC TGTCGTGTGC AGCAAGCGGT 481TTCAACATCA AAGATACCTA CATCCACTGG GTTCGTCAGG CGCCGGGCAA AGGTCTGGAA 541TGGGTCGCCC GCATTTACCC GACCAATGGC TATACGCGTT ACGCAGATAG CGTGAAAGGT 601CGCTTTACCA TCTCTGCGGA CACCAGTAAA AACACGGCCT ATCTGCAGAT GAATAGCCTG 661CGTGCGGAAG ATACGGCCGT TTATTACTGC TCTCGCTGGG GTGGCGATGG CTTCTATGCT 721ATGGACTACT GGGGCCAGGG TACCCTGGTG ACGGTTTCAT CGGGTCAGCC GCGTGAACCG 781CAAGTGTATA CCCTGCCGCC GTCACGCGAT GAACTGACGA AAAACCAGGT GTCGCTGACG 841TGTCTGGTTA AAGGCTTTTA CCCGAGCGAC ATCGCGGTTG AATGGGAATC TAATGGTCAA 901CCGGAAAACA ATTATAAAAC CACGCCGCCG GTCCTGGATA GTGACGGCTC CTTTTTCCTG 961TACAGTAAAC TGACCGTGGA TAAATCCCGT TGGCAGCAGG GTAACGTCTT CTCGTGTAGC 1021GTGATGCATG AAGCCCTGCA TAATCACTAT ACCCAGAAAT CTCTGAGTCT GTCCCCGGGC 1081AAAGGTTCAA CGTCGGGTGG CGGTTCCGGC GGTGGCTCAG GTGGCGGTGG CAGCTCTGGC 1141CAACCGCGCG AACCGCAGGT TTACACCCTG CCGCCGAGCC GTGACGAACT GACCAAAAAC 1201CAAGTCAGCC TGACGTGCCT GGTGAAAGGC TTTTACCCGA GTGACATTGC AGTTGAATGG 1261GAATCCAATG GTCAGCCGGA AAATAACTAC AAAACGACGC CGCCGGTTCT GGATTCAGAC 1321GGCTCGTTTT TCCTGTACTC AAAACTGACC GTCGATAAAT CGCGCTGGCA ACAGGGTAAC 1381GTTTTCAGCT GCTCTGTCAT GCACGAAGCC CTGCACAACC ATTATACCCA GAAAAGTCTG 1441TCCCTGTCAC CGGGCAAAGA AGTGCAGCTG GTTGAATCTG GTGGCGGTCT GGTGCAACCG 1501GGCGGTTCGC TGCGTCTGAG CTGTGCAGCT TCTGGCTTTA ATATTAAAGA CACGTACATC 1561CACTGGGTGC GTCAGGCACC GGGTAAAGGC CTGGAATGGG TTGCTCGTAT CTATCCGACG 1621AACGGTTATA CGCGTTACGC CGATAGCGTC AAAGGCCGTT TTACCATCAG TGCAGACACC 1681TCCAAAAACA CGGCTTATCT GCAGATGAAT AGTCTGCGTG CAGAAGATAC CGCTGTTTAT 1741TACTGCAGCC GCTGGGGCGG TGATGGCTTC TATGCAATGG ATTATTGGGG TCAAGGTACC 1801CTGGTCACCG TGAGTTCCGG TTCGACCAGC GGCGGTGGCT CAGGTGGCGG TTCGGGCGGT 1861GGCGGTTCAT CGGACATTCA GATGACGCAA AGCCCGAGCT CTCTGTCTGC GAGTGTTGGC 1921GATCGTGTCA CCATCACGTG TCGCGCCTCT CAGGACGTGA ATACCGCAGT TGCTTGGTAC 1981CAACAAAAAC CGGGCAAAGC ACCGAAACTG CTGATTTACT CCGCTTCATT CCTGTACAGC 2041GGTGTGCCGT CTCGTTTTTC GGGCAGCCGC TCTGGTACCG ATTTCACCCT GACGATTAGT 2101TCCCTGCAAC CGGAAGATTT CGCCACCTAC TACTGCCAGC AACACTATAC GACCCCGCCG 2161ACGTTTGGTC AGGGCACGAA AGTGGAAATT AAAAACCGTG TGCGTCGCAG CAAATTTGCG 2221AAATTCGCCA AAAAATTTGC AAAATTCGCT AAAAAATTTG CGAAATAATG AAAGCTT5. scFv-C_(H)3-NRVRRS-(KLAKLAK)₂KLAK: (SEQ ID NO.: 80) 1CATATGCATC ACCACCACCA CCACGACGAC GACGACAAAG ATATTCAAAT GACCCAGTCC 61CCGAGCAGCC TGAGTGCCTC CGTTGGCGAC CGCGTGACCA TTACGTGCCG TGCGAGCCAG 121GATGTCAACA CCGCGGTGGC CTGGTATCAG CAAAAACCGG GCAAAGCGCC GAAACTGCTG 181ATCTATTCAG CCTCGTTTCT GTACAGCGGT GTTCCGTCTC GTTTCAGCGG CTCTCGCAGT 241GGTACCGATT TTACCCTGAC GATTAGCTCT CTGCAGCCGG AAGACTTTGC GACGTATTAC 301TGCCAGCAAC ATTACACCAC GCCGCCGACC TTCGGCCAGG GTACGAAAGT GGAAATCAAA 361GGTTCCACCT CAGGCGGTGG CAGTGGTGGC GGTTCCGGCG GTGGCGGTAG TTCCGAAGTT 421CAGCTGGTCG AAAGTGGCGG TGGCCTGGTT CAACCGGGTG GCTCACTGCG TCTGTCGTGT 481GCAGCAAGCG GTTTCAACAT CAAAGATACC TACATCCACT GGGTTCGTCA GGCGCCGGGC 541AAAGGTCTGG AATGGGTCGC CCGCATTTAC CCGACCAATG GCTATACGCG TTACGCAGAT 601AGCGTGAAAG GTCGCTTTAC CATCTCTGCG GACACCAGTA AAAACACGGC CTATCTGCAG 661ATGAATAGCC TGCGTGCGGA AGATACGGCC GTTTATTACT GCTCTCGCTG GGGTGGCGAT 721GGCTTCTATG CTATGGACTA CTGGGGCCAG GGTACCCTGG TGACGGTTTC ATCGGGTCAG 781CCGCGTGAAC CGCAAGTGTA TACCCTGCCG CCGTCACGCG ATGAACTGAC GAAAAACCAG 841GTGTCGCTGA CGTGTCTGGT TAAAGGCTTT TACCCGAGCG ACATCGCGGT TGAATGGGAA 901TCTAATGGTC AACCGGAAAA CAATTATAAA ACCACGCCGC CGGTCCTGGA TAGTGACGGC 961TCCTTTTTCC TGTACAGTAA ACTGACCGTG GATAAATCCC GTTGGCAGCA GGGTAACGTC 1021TTCTCGTGTA GCGTGATGCA TGAAGCCCTG CATAATCACT ATACCCAGAA ATCTCTGAGT 1081CTGTCCCCGG GCAAAGGTTC AACGTCGGGT GGCGGTTCCG GCGGTGGCTC AGGTGGCGGT 1141GGCAGCTCTG GCCAACCGCG CGAACCGCAG GTTTACACCC TGCCGCCGAG CCGTGACGAA 1201CTGACCAAAA ACCAAGTCAG CCTGACGTGC CTGGTGAAAG GCTTTTACCC GAGTGACATT 1261GCAGTTGAAT GGGAATCCAA TGGTCAGCCG GAAAATAACT ACAAAACGAC GCCGCCGGTT 1321CTGGATTCAG ACGGCTCGTT TTTCCTGTAC TCAAAACTGA CCGTCGATAA ATCGCGCTGG 1381CAACAGGGTA ACGTTTTCAG CTGCTCTGTC ATGCACGAAG CCCTGCACAA CCATTATACC 1441CAGAAAAGTC TGTCCCTGTC ACCGGGCAAA GAAGTGCAGC TGGTTGAATC TGGTGGCGGT 1501CTGGTGCAAC CGGGCGGTTC GCTGCGTCTG AGCTGTGCAG CTTCTGGCTT TAATATTAAA 1561GACACGTACA TCCACTGGGT GCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTTGCTCGT 1621ATCTATCCGA CGAACGGTTA TACGCGTTAC GCCGATAGCG TCAAAGGCCG TTTTACCATC 1681AGTGCAGACA CCTCCAAAAA CACGGCTTAT CTGCAGATGA ATAGTCTGCG TGCAGAAGAT 1741ACCGCTGTTT ATTACTGCAG CCGCTGGGGC GGTGATGGCT TCTATGCAAT GGATTATTGG 1801GGTCAAGGTA CCCTGGTCAC CGTGAGTTCC GGTTCGACCA GCGGCGGTGG CTCAGGTGGC 1861GGTTCGGGCG GTGGCGGTTC ATCGGACATT CAGATGACGC AAAGCCCGAG CTCTCTGTCT 1921GCGAGTGTTG GCGATCGTGT CACCATCACG TGTCGCGCCT CTCAGGACGT GAATACCGCA 1981GTTGCTTGGT ACCAACAAAA ACCGGGCAAA GCACCGAAAC TGCTGATTTA CTCCGCTTCA 2041TTCCTGTACA GCGGTGTGCC GTCTCGTTTT TCGGGCAGCC GCTCTGGTAC CGATTTCACC 2101CTGACGATTA GTTCCCTGCA ACCGGAAGAT TTCGCCACCT ACTACTGCCA GCAACACTAT 2161ACGACCCCGC CGACGTTTGG TCAGGGCACG AAAGTGGAAA TTAAAAACCG TGTGCGTCGC 2221AGCAAACTGG CGAAACTGGC CAAAAAACTG GCAAAACTGG CTAAAAAACT GGCGAAATAA 2281TGAAAGCTT 6. Phor18-scFv-C_(H)3-Phor18: (SEQ ID NO.: 81) 1CATATGGAAA ATCTGTATTT CCAAGGTAAA TTTGCGAAAT TCGCCAAAAA ATTCGCAAAA 61TTCGCGAAAA AATTCGCGAA 

GATATTCAA ATGACCCAGT CCCCGAGCAG CCTGAGTGCC 121TCCGTTGGCG ACCGCGTGAC CATTACGTGC CGTGCGAGCC AGGATGTCAA CACCGCGGTG 181GCCTGGTATC AGCAAAAACC GGGCAAAGCG CCGAAACTGC TGATCTATTC AGCCTCGTTT 241CTGTACAGCG GTGTTCCGTC TCGTTTCAGC GGCTCTCGCA GTGGTACCGA TTTTACCCTG 301ACGATTAGCT CTCTGCAGCC GGAAGACTTT GCGACGTATT ACTGCCAGCA ACATTACACC 361ACGCCGCCGA CCTTCGGCCA GGGTACGAAA GTGGAAATCA AAGGTTCCAC CTCAGGCGGT 421GGCAGTGGTG GCGGTTCCGG CGGTGGCGGT AGTTCCGAAG TTCAGCTGGT CGAAAGTGGC 481GGTGGCCTGG TTCAACCGGG TGGCTCACTG CGTCTGTCGT GTGCAGCAAG CGGTTTCAAC 541ATCAAAGATA CCTACATCCA CTGGGTTCGT CAGGCGCCGG GCAAAGGTCT GGAATGGGTC 601GCCCGCATTT ACCCGACCAA TGGCTATACG CGTTACGCAG ATAGCGTGAA AGGTCGCTTT 661ACCATCTCTG CGGACACCAG TAAAAACACG GCCTATCTGC AGATGAATAG CCTGCGTGCG 721GAAGATACGG CCGTTTATTA CTGCTCTCGC TGGGGTGGCG ATGGCTTCTA TGCTATGGAC 781TACTGGGGCC AGGGTACCCT GGTGACGGTT TCATCGGGTC AGCCGCGTGA ACCGCAAGTG 841TATACCCTGC CGCCGTCACG CGATGAACTG ACGAAAAACC AGGTGTCGCT GACGTGTCTG 901GTTAAAGGCT TTTACCCGAG CGACATCGCG GTTGAATGGG AATCTAATGG TCAACCGGAA 961AACAATTATA AAACCACGCC GCCGGTCCTG GATAGTGACG GCTCCTTTTT CCTGTACAGT 1021AAACTGACCG TGGATAAATC CCGTTGGCAG CAGGGTAACG TCTTCTCGTG TAGCGTGATG 1081CATGAAGCCC TGCATAATCA CTATACCCAG AAATCTCTGA GTCTGTCCCC GGGCAAAGGT 1141TCAACGTCGG GTGGCGGTTC CGGCGGTGGC TCAGGTGGCG GTGGCAGCTC TGGCCAACCG 1201CGCGAACCGC AGGTTTACAC CCTGCCGCCG AGCCGTGACG AACTGACCAA AAACCAAGTC 1261AGCCTGACGT GCCTGGTGAA AGGCTTTTAC CCGAGTGACA TTGCAGTTGA ATGGGAATCC 1321AATGGTCAGC CGGAAAATAA CTACAAAACG ACGCCGCCGG TTCTGGATTC AGACGGCTCG 1381TTTTTCCTGT ACTCAAAACT GACCGTCGAT AAATCGCGCT GGCAACAGGG TAACGTTTTC 1441AGCTGCTCTG TCATGCACGA AGCCCTGCAC AACCATTATA CCCAGAAAAG TCTGTCCCTG 1501TCACCGGGCA AAGAAGTGCA GCTGGTTGAA TCTGGTGGCG GTCTGGTGCA ACCGGGCGGT 1561TCGCTGCGTC TGAGCTGTGC AGCTTCTGGC TTTAATATTA AAGACACGTA CATCCACTGG 1621GTGCGTCAGG CACCGGGTAA AGGCCTGGAA TGGGTTGCTC GTATCTATCC GACGAACGGT 1681TATACGCGTT ACGCCGATAG CGTCAAAGGC CGTTTTACCA TCAGTGCAGA CACCTCCAAA 1741AACACGGCTT ATCTGCAGAT GAATAGTCTG CGTGCAGAAG ATACCGCTGT TTATTACTGC 1801AGCCGCTGGG GCGGTGATGG CTTCTATGCA ATGGATTATT GGGGTCAAGG TACCCTGGTC 1861ACCGTGAGTT CCGGTTCGAC CAGCGGCGGT GGCTCAGGTG GCGGTTCGGG CGGTGGCGGT 1921TCATCGGACA TTCAGATGAC GCAAAGCCCG AGCTCTCTGT CTGCGAGTGT TGGCGATCGT 1981GTCACCATCA CGTGTCGCGC CTCTCAGGAC GTGAATACCG CAGTTGCTTG GTACCAACAA 2041AAACCGGGCA AAGCACCGAA ACTGCTGATT TACTCCGCTT CATTCCTGTA CAGCGGTGTG 2101CCGTCTCGTT TTTCGGGCAG CCGCTCTGGT ACCGATTTCA CCCTGACGAT TAGTTCCCTG 2161CAACCGGAAG ATTTCGCCAC CTACTACTGC CAGCAACACT ATACGACCCC GCCGACGTTT 2221GGTCAGGGCA CGAAAGTGGA AATTAAAAAA TTTGCGAAAT TCGCCAAAAA ATTCGCAAAA 2281TTCGCGAAAA AATTCGCGAA ATAATGAAAG CTT

Chemical Conjugation of Phor18 to a Monoclonal Anti-Her2/Neu AntibodyIRG1 (MAb):

Purified antibody in phosphate buffered saline (PBS) is concentrated toa concentration of approximately 2 mg/mL. A 20 mM solution ofN-succinidyl-3-(2-pyridylothio)propionate (SPDP) is freshly prepared indimethylsulfoxide (DMSO), and added to the antibody solution in 20-foldexcess. The mixture is incubated at room temperature for about 30minutes to produce the antibody-linker intermediate. Excess unreactedSPDP is removed by size exclusion. The cytotoxic molecule containingcysteine is thoroughly reduced by reaction with a 10-fold excess ofreductacryl reagent before mixing in 10-fold excess with theantibody-linker construct. The reaction is allowed to incubate at roomtemperature for 18 hours, then desalted to remove unreacted cytotoxinmolecule. The solution is filter-sterilized before storage.

In Vitro:

In vitro cytotoxicity studies were performed to determine thecytotoxicity of the recombinant antibody preparations (conjugated andunconjugated) and lytic peptide, Phor18, was used in controlincubations. Cells were prepared in 96 well plates using 2,000cells/well and were allowed to attach for 48 hours. Phor18 inlyophilized form was freshly dissolved in saline and added into themulti-well plates at increasing concentrations of 0, 0.00001, 0.0001,0.001, 0.01, 0.1, 1, 10, 25, and 100 μM. The Her2/neu-antibody-Phor18conjugates (Phor18-scFv-C_(H)3-Phor18, scFv-C_(H)3-GS-Phor18,scFv-C_(H)3-NRVRRS (SEQ ID NO.:75)-Phor18, theHer2/neu-antibody-(KLAKLAK)₂KLAK (SEQ ID NO.:74)(scFv-C_(H)3-GS-(KLAKLAK)₂KLAK (SEQ ID NO.:74), and scFv-C_(H)3-NRVRRS(SEQ ID NO.:75)-(KLAKLAK)₂KLAK) (SEQ ID NO.:74), or scFv-C_(H)3-receptorantibody (naked) in Tris/HCL-buffer were diluted with saline and addedto cells at increasing concentrations of 0, 0.0012, 0.012, 0.12, 1.2,6.0, 12.0, 120, 360 and 720 nM. Incubations were conducted for 48 h at37° C. Cell viability was determined using formazan conversion assays(MTT assays). Controls contained USP saline or 0.1% TritonX-100™ asreference for 0 and 100% cell death, respectively. All data wereprocessed and analyzed using Graph Pad Prizm 4™ software (Graph PadPrizm, Inc).

Example 3

This example describes studies indicating that anti-Her2-Phor18 antibodyconjugate killed Her2 expressing breast cancer cells.

As shown in Table 2, the anti-Her2-Phor18 antibody conjugates(Phor18-scFv-C_(H)3-Phor18, scFv-C_(H)3-GS-Phor18, scFv-C_(H)3-NRVRRS(SEQ ID NO.:75)-Phor18) killed Her2/neu positive human breast cancerSKBR-3 and ovarian cancer SKOV-3 cell lines by 48 hours, whereas theHer2/neu negative human breast cancer MDA-MB-231 cell line was notkilled. Evidence of cytotoxicity was observed microscopically at asearly as 24 hours of incubation. As expected, unconjugated Phor18 showedonly modest cytotoxicity.

The HER2/neu antibody conjugated to the Phor18 was significantly morecytotoxic than antibody conjugated to the lytic peptide(KLAKLAK)₂KLAK(SEQ ID NO.:74). (FIG. 1, Table 2). The Her2/neu negativeMDA-MB-231 cells were not killed by any of the recombinantantibody-lytic peptide conjugates indicating that the cytotoxicity ofthe antibodies was mediated via Her2/neu receptors. The “naked”(unconjugated) antibody (scFv-C_(H)3) was not cytotoxic in all 3 celllines indicating that the cell-killing properties of the antibody-lyticpeptide conjugates were due to the presence of lytic peptide payload andsequence of the lytic peptide. Again, as expected, unconjugated Phor18showed very minimal non-specific cytotoxicity in all cell lines (Table2).

TABLE 2In vitro cytotoxicity of anti-Her2-Phor18 antibody conjugates (scFv-C_(H)3-Phor18 and -scFv-C_(H)3-(KLAKLAK)₂KLAK (SEQ ID NO.: 74) conjugates, Her2/neu scFv-C_(H)3 and unconjugated Phor18in Her2/neu receptor positive SKOV-3, SKBR-3 and Her2/neu receptor negative MDA-MB-231 cancercells. Values are IC₅₀ expressed in nM. IC₅₀ [nM] IC₅₀ [nM] IC₅₀ [nM]Recombinant Antibody Conjugate SKOV-3 SKBR-3 MDA-MB-231Phor18-scFv-C_(H)3-Phor18 44.33 ± 9.2 51.56 ± 6.1 >1000scFv-C_(H)3-GS-Phor18    27 ± 2.5    30 ± 1.9 >1000scFv-C_(H)3-GS-(KLAKLAK)₂KLAK   235 ± 6.5   246 ± 41 >1000scFv-C_(H)3-NRVRRS-Phor18  29.3 ± 3.5  76.3 ± 16 >1000scFv-C_(H)3-NRVRRS-(KLAKLAK)₂KLAK   247 ± 40.5   338 ± 8.7 >1000Her2/neu scFv-C_(H)3 >1000 >1000 >1000 Phor18 18,180 11,455 9,258

The results indicate that recombinantly produced Her2 antibodyscFv-C_(H)3-Phor18 and Her2 antibody scFv-C_(H)3-(KLAKLAK)₂KLAK (SEQ IDNO.:74) conjugates are active in the nanomolar range against Her2/neureceptor expressing cell lines. The unconjugated antibody or free lyticpeptide (Phor18) were without effect indicating that conjugation oflytic peptides to ligands (e.g., antibodies) that bind to Her2/neureceptor to enhances cell cytotoxic potency.

Example 4

This example includes a description of in vitro cytotoxicity studies ofrecombinantly produced antibody to Her-2 receptor conjugated to lyticpeptide, Phor18 (KFAKFAK KFAKFAK KFAK (SEQ ID NO.:4))(scFv-C_(H)3-GS-Phor18), and a chemically conjugated MAb-Phor18conjugate against Her2 positive ovarian cancer cell line SKOV-3.

Cells were prepared in 96 well plates using 5,000 cells/well and wereallowed to attach for 48 hours. MAb-Phor18, scFv-C_(H)3-GS-Phor18,scFv-C_(H)3 were diluted in saline and added at increasingconcentrations of 0, 0.0012, 0.012, 0.12, 1.2, 6.0, 12, 120, 360 and 720nM, N=8 data points per concentration. Incubations were conducted for 24h at 37° C. Cell viability was determined using formazan conversionassays (MTT assays). Controls contained USP saline or 0.1% TritonX-100™as reference for 0 and 100% cell death, respectively.

Data were processed and analyzed using Graph Pad Prizm 4™ software(Graph Pad Prizm, Inc). Statistical analysis for significance wasdetermined by a two-tailed Student's T-test. The whole MAb-Phor18resulted in IC₅₀ values of 60.53±3.8 nM and scFv-C_(H3)-GS-Phor18 was59.8±3.8 nM. The “naked” (unconjugated) antibodies (MAb and scFv-C_(H)3)were not cytotoxic. In vitro chemically linked HER2 antibody(MAb-Phor18) and recombinant Phor18 conjugate (scFv-C_(H3)-GS-Phor18)showed similar toxicity to SKOV-3 cells, whereas the naked recombinantantibody (scFv-C_(H)3) was not toxic.

Example 5

This example describes an in vivo study in a mouse xenograft model ofhuman ovarian cancer with various doses of anti-Her2-Phor18 antibodyconjugates (scFv-C_(H3)-GS-Phor18, MAb-Phor18), naked whole antibody(MAb) and naked recombinant antibody (scFv-C_(H)3) treatments.

Female Nu/Nu mice were injected subcutaneously with a SKOV-3/Matrigelsuspension (4×10⁶ cells). Tumor weights from mice that were killed onday 42 served as baseline. In brief, treatment started on day 43 aftertumor cell injection on tumors of median tumor volume of 130.3±10.25 mm³and continued on days 47, 50, 54, 57 and 60 as a single bolus injectioninto the lateral tail vein.

During the entire study tumor volumes were measured twice per week andbody weights were determined. Final necropsy was conducted on day 64after tumor cell injection where tumors were excised, weighed and fixedin formalin for histological evaluation.

Treatments were: saline control, whole naked monoclonalanti-Her2-antibody, MAb, (3 mg/kg), recombinant naked-Her2-antibody(scFv-C_(H)3) (3 mg/kg), scFv-C_(H)3-GS-Phor18 (0.3 and 3 mg/kg),MAb-Phor18 (0.3 and 3 mg/kg). Tumors from mice sacrificed at treatmentstart underwent immunohistochemistry evaluation of Her2/neu receptors.Each group consisted of 8-9 mice.

All groups of mice tolerated the injections well. No mice died as aconsequence of injection.

The effect of antibody conjugated Phor18 injections and naked antibodieson the primary tumors (volume and tumor weights, FIGS. 2A, 2B, and 3)and body weight is illustrated in FIG. 4. FIGS. 2A and 2B show mediantumor volumes during the course of the study and mean tumor weights onday 64 for each individual treatment group for saline controls, and micetreated with MAb (naked) (3 mg/kg), scFv-C_(H)3 (3 mg/kg),scFv-C_(H)3-GS-Phor18 (0.3 and 3 mg/kg), MAb-Phor18 (0.3 and 3 mg/kg).

Tumor volumes and weights decreased significantly in all animals treatedwith 3 mg/kg MAb-Phor18 chemically linked (p<0.04) or recombinantlyproduced scFv-CH₃-Phor18 conjugates (p<0.02). Naked MAb or scFv-C_(H)3were not decreasing tumor volumes or tumor weights compared to salinecontrols at doses of 3 mg/kg (FIGS. 2A, 2B, and 3). Statistical analysiswas conducted in Graphpad prizm 4 using the Wilcoxon signed rank test.Body weights were stable in all treatment groups and control animals(FIG. 4).

Example 6

This example describes in vitro cytotoxicity studies of recombinantlyproduced antibody to Her-2 receptor conjugated to lytic peptide, Phor18(KFAKFAK KFAKFAK KFAK(SEQ ID NO.:4)) against ovarian cancer cells.

scFv-C_(H)2-C_(H3)-GS-Phor18 (one molecule of Phor18 joined to theantibody at the C-terminus by GS linker, consisting of V_(L)-Glinker-V_(H)-C_(H)2-C_(H)3-G linker-C_(H)3-C_(H)2-V_(H)-Glinker-V_(L)-GS-(Phor18). Cytotoxicity was compared to a naked antibody(scFv-C_(H)2-C_(H)3; antibody without a lytic peptide) in Her-2 receptorpositive cells (SKOV-3, human ovarian cancer cells).

Materials: Recombinant DNA technique was used to synthesize anti-Her2antibody as an scFv-C_(H)2-C_(H)3 antibody in Escherichia coli. Theantibody (Olafsen T. et al Protein Engineering, Design & Selection 17,315-323, 2004) was conjugated via a peptide linker to either Phor18 andanalyzed for cytotoxicity in vitro. The plasmid was acquired throughgene codon optimization. The gene was synthesized with a N-His tagsequence and the plasmid was subcloned into an E. coli bacteriaexpression vector pUC57. After expression optimization and evaluationthe His-tag product was selected and 1 L of the bacteria expressionproduct was purified in a one-step affinity purification. The amino acidsequence of for each construct is described in Table 3.

TABLE 3Amino Acid sequence for the production of each recombinant antibody,(A) scFv-C_(H)2- C_(H)3; and antibody conjugate (B) scFv-C_(H)2-C_(H)3-GS-Phor18.A) scFv-C_(H)2-C_(H)3; and antibody conjugate: (SEQ ID NO.: 82)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS        70         80         90        100        110        120RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKGST SGGGSGGGSG       130        140        150        160        170        180GGGSSEVQLV ESGGGLVQPG GSLRLSCAAS GFNIKDTYIH WVRQAPGKGL EWVARIYPTN       190        200        210        220        230        240GYTRYADSVK GRFTISADTS KNTAYLQMNS LRAEDTAVYY CSRWGGDGFY AMDYWGQGTL       250        260        270        280        290        300VTVSSPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV       310        320        330        340        350        360EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ       370        380        390        400        410        420PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG       430        440        450        460        470        480SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKGSTSG GGSGGGSGGG       490        500        510        520        530        540GSSGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV       550        560        570        580        590        600LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK PCPAPELLGG       610        620        630        640        650        660PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN       670        680        690        700        710        720STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKEVQLVES GGGLVQPGGS       730        740        750        760        770        780LRLSCAASGF NIKDTYIHWV RQAPGKGLEW VARIYPTNGY TRYADSVKGR FTISADTSKN       790        800        810        820        830        840TAYLQMNSLR AEDTAVYYCS RWGGDGFYAM DYWGQGTLVT VSSGSTSGGG SGGGSGGGGS       850        860        870        880        890        900SDIQMTQSPS SLSASVGDRV TITCRASQDV NTAVAWYQQK PGKAPKLLIY SASFLYSGVP       910        920        930        940SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ QHYTTPPTFG QGTKVEIKB) scFv-C_(H)2-C_(H)3-GS-Phor18: (SEQ ID NO.: 83)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS        70         80         90        100        110        120RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKGST SGGGSGGGSG       130        140        150        160        170        180GGGSSEVQLV ESGGGLVQPG GSLRLSCAAS GFNIKDTYIH WVRQAPGKGL EWVARIYPTN       190        200        210        220        230        240GYTRYADSVK GRFTISADTS KNTAYLQMNS LRAEDTAVYY CSRWGGDGFY AMDYWGQGTL       250        260        270        280        290        300VTVSSPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV       310        320        330        340        350        360EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ       370        380        390        400        410        420PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG       430        440        450        460        470        480SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKGSTSG GGSGGGSGGG       490        500        510        520        530        540GSSGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV       550        560        570        580        590        600LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK PCPAPELLGG       610        620        630        640        650        660PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN       670        680        690        700        710        720STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKEVQLVES GGGLVQPGGS       730        740        750        760        770        780LRLSCAASGF NIKDTYIHWV RQAPGKGLEW VARIYPTNGY TRYADSVKGR FTISADTSKN       790        800        810        820        830        840TAYLQMNSLR AEDTAVYYCS RWGGDGFYAM DYWGQGTLVT VSSGSTSGGG SGGGSGGGGS       850        860        870        880        890        900SDIQMTQSPS SLSASVGDRV TITCRASQDV NTAVAWYQQK PGKAPKLLIY SASFLYSGVP       910        920        930        940        950        960SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ QHYTTPPTFG QGTKVEIKGS KFAKFAKKFAKFAKKFAK

In vitro cytotoxicity studies were performed to determine cytotoxicityof the recombinant antibody preparations (scFv-C_(H)2-C_(H)3,scFv-C_(H)3, and scFv-C_(H)2-C_(H)3-GS-Phor18, scFv-C_(H)3-GS-Phor18).Her-2 receptor positive SKOV-3 cells were prepared in 96 well platesusing 2,000 cells/well and were allowed to attach for 48 hours. TheHer2-antibody-Phor18 conjugates (scFv-C_(H)2-C_(H)3-GS-Phor18,scFv-C_(H)3 GS-Phor18) or the naked antibodies (scFv-C_(H)2-C_(H)3,scFv-C_(H)3) in Tris/HCL-buffer were diluted with saline and added tocells at increasing concentrations of 0, 0.0012, 0.012, 0.12, 1.2, 6.0,12.0, 120, 360 and 720 nM. Incubations were conducted for 48 h at 37° C.Cell viability was determined using Cell Titer Glo luminescent cellviability assay (Promega). Controls contained USP saline or 0.1%TritonX-100™ as reference for 0 and 100% cell death, respectively. Alldata were processed and analyzed using Graph Pad Prizm 4™ software(Graph Pad Prizm, Inc).

The Her2 antibody (scFv-C_(H)2-C_(H)3, scFv-C_(H)3) conjugated to thePhor18 resulted in IC₅₀ values of 53.7±0.63 nM for scFv-C_(H)3-Phor18and 56.7±0.92 nM for scFv-CH₂—CH₃—Fv-Phor18. The “naked” (unconjugated)antibodies, scFv-C_(H)2-C_(H)3, scFv-C_(H)3, were not cytotoxic. Invitro recombinantly Phor18 conjugates show similar toxicity to SKOV-3cells.

Example 7

This example includes a description of the construction and expressionof whole anti-Her2-IgG1-Phor18 antibody conjugates with defined locationlytic domain (Phor18), and specified numbers of 2, 4 and 6 Phor18 lyticdomains per antibody in a mammalian expression system. These conjugatesare also referred to as antibody-drug conjugates (ADC).

Recombinant expression of whole IgG1 antibody-Phor18 (KFAKFAKKFAKFAKKFAK(SEQ ID NO.:4)) conjugates in a mammalian system (CHO cells) wasconducted using two different secretion signal sequences: a proprietarysecretion signal sequence for antibody heavy and light chains, and humanIgG kappa-light chain secretion signal. The expressed anti-Her2 IgG1antibody (humanized variable light and heavy domains regions to Her-2receptor) and the various antibody-Phor18 conjugates with stoichiometricratios of Phor18: AB of 2, 4 and 6 were characterized using SDS PAGE,Western blot analyses, and surface Plasmon resonance (selected ADCs).

Yield, purity and cytotoxicity of recombinantly produced antibodies (asa full IgG1 antibody) with Heavy (H) or Light (L) chain C-terminal- orN-terminal-Phor18 conjugation was analyzed. Two, 4 and 6 molecules oflytic domains (Phor18) conjugated to whole antibody molecule wereexpressed. The amino acid sequences of the “unconjugated anti-Her2antibody, anti-Her2 antibody heavy (H) and light (L) chains are shown inTable 4.

TABLE 4Anti-Her2/neu antibody amino acid sequence from Drugbank.ca DB00072.There was one additional amino acid in the C_(H)1 domain of the Anti-Her2antibody in comparison to human IgG1 (underlined).Anti-HER2/neu Light chain (naked L1): (SEQ ID NO.: 84)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS        70         80         90        100        110        120RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP       130        140        150        160        170        180SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSED STYSLSSTLT       190        200        210 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECAnti-HER2/neu Heavy chain 2 (naked H1): (SEQ ID NO.: 85)        10         20         30         40         50         60EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY        70         80         90        100        110        120ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS       130        140        150        160        170        180ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS       190        200        210        220        230        240GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP PKSCDKTHTC PPCPAPELLG       250        260        270        280        290        300GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY       310        320        330        340        350        360NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD       370        380        390        400        410        420ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR       430        440        450 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

The amino acid sequences of antibody-lytic peptide conjugate heavy (H)and light (L) chains are shown in Table 5. Gene synthesis was conductedat Genewiz, Inc (South Plainfield, N.J.) using preferred codon usage forChinese Hamster Ovary cells. The transcripts were ligated into the pUC57bacterial plasmid.

TABLE 5Amino acid sequences of lytic-peptide (Phor18, KFAKFAKKFAKFAKKFAK(SEQ ID NO.: 4))-antibody heavy and light chain conjugatesPhor18-V_(L) Light Chain (L2): (SEQ ID NO.: 86)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP        70         80         90        100        110        120GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ       130        140        150        160        170        180GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ       190        200        210        220        230ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECC_(L)-Phor18 Light Chain (L3): (SEQ ID NO.: 87)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS        70         80         90        100        110        120RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP       130        140        150        160        170        180SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSED STYSLSSTLT       190        200        210        220        230 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECGSKFAK FAKKFAKFAK KFAKAPhor18-V_(L)-C_(L)-Phor18 Light Chain (L4): (SEQ ID NO.: 88)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP        70         80         90        100        110        120GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ       130        140        150        160        170        180GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ       190        200        210        220        230        240ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECGSKFAK       250 FAKKFAKFAK KFAKAPhor18-V_(H) Heavy Chain (H2): (SEQ ID NO.: 89)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA        70         80         90        100        110        120PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG       130        140        150        160        170        180GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS       190        200        210        220        230        240WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP       250        260        270        280        290        300PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN       310        320        330        340        350        360WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI       370        380        390        400        410        420SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP       430        440        450        460        470VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KC_(H)3-Phor18 Heavy Chain (H3): (SEQ ID NO.: 90)        10         20         30         40         50         60EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY        70         80         90        100        110        120ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS       130        140        150        160        170        180ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS       190        200        210        220        230        240GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP PKSCDKTHTC PPCPAPELLG       250        260        270        280        290        300GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY       310        320        330        340        350        360NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD       370        380        390        400        410        420ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR       430        440        450        460        470WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGSKFAKFAK KFAKFAKKFA KAPhor18-V_(H)-C_(H)3-Phor18 Heavy Chain (H4): (SEQ ID NO.: 91)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA        70         80         90        100        110        120PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG       130        140        150        160        170        180GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS       190        200        210        220        230        240WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP       250        260        270        280        290        300PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN       310        320        330        340        350        360WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI       370        380        390        400        410        420SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP       430        440        450        460        470        480VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGSKFAKFAK       490 KFAKFAKKFA KA

Heavy (H) and light (L) chain transcripts were synthesized out ofGenewiz pUC57 plasmids using PCR with primers containing 5′ EcoR1 and 3′Xba1 restriction sites for directional ligation into the multiplecloning site of the pCMVTnT mammalian expression plasmid (FIG. 5,Promega, Madison, Wis., L5620, lot 14524919). Each transcript included aKozak consensus sequence and a secretion signal at the 5′ end.

Anti-Her2/neu antibody was produced by Lonza Inc. (Cambridge, UK). AChinese Hamster Ovary (CHO) cell mammalian expression system was used toproduce naked (i.e., no lytic domain) anti-Her2/neu IgG1, and ADCshaving a lytic domain (Phor18) at defined locations (N- or C-terminal)and in ratios of 2, 4 and 6 lytic domains per whole antibody.

In brief, Free-style CHO Suspension Cell Expression System (InvitrogenLife Sciences, Carlsbad, Calif., cat# K9000-20) was used to grow FS-CHOcells (according to manufacturer's directions) in FreeStyle CHOexpression medium supplemented with 8 mM glutamax and 5 ml/Lpenicillin/streptomycin. Cells were thawed into growth medium withoutpenicillin/streptomycin that was prewarmed to 37° C. and equilibrated inan 8% CO₂ atmosphere and grown in 30 ml in shaker flasks at 125-135 rpm.Cell density was kept at or below 1×10⁶ cells/ml to avoid clumping.

FS-CHO cells were transfected according to the Invitrogen protocol. FSCHO cells were expanded for 7 or more days after thawing. Cells weredoubling every 24 h. The day before transfection, clumps were removedand cells were pelleted and resuspended in P/S-free medium at 5×10⁵/ml.On the day of transfection, cells were adjusted to 9×10⁵/ml if necessaryand viability was close to 99%. Each 500 ml spinner flask (VWR, cat# PBV125) of 180 ml cells was transfected with 180 μg of total plasmid DNAmixed with 180 μl of FSMax transfection reagent (Invitrogen,cat#16447-100). Cells were swirled rapidly while adding DNA mixtureslowly. Ratios of H:L chains analyzed were 3:2, 1:1, and 2:3.

ADC secreted into the cell medium, harvested on day 4 to day 6 aftertransfection, was purified using protein A columns. Approximately 0.25ml of protein A resin (Genscript L00210, capacity >20 mg IgG per mlresin) was used to isolate secreted ADCs from the FS CHO medium usingthe Genscript product protocol and buffer descriptions provided. ADC waseluted once with 0.5 ml low pH elution buffer of 0.1M glycine pH 2.5 andeluate was reapplied to the column and collected a second time. pH wasadjusted to 6.5 with 1M Tris pH 8.

To confirm the presence of protein, SDS-PAGE (4-15% TGX gels, Bio-RadLabs, Hercules, Calif., cat#456-1084) analyses was used. ADCs separatedon gels were silver-stained (Sigma-Aldrich, St Louis, Mo., Prot Sillkit). The anti-Phor18 rabbit polyclonal was from Covance (Denver, Pa.,cat#338983), and for detection an anti-rabbit-HRP was used(cat#111-035-046, Jackson ImmunoResearch, Philadelphia, Pa.). Detectionof ADCs was conducted with an anti-human IgG from Jackson ImmunoResearch(cat#109-035-088). The presence of the (L) light chain was confirmed onreduced ADCs with HRP-mouse anti-human kappa (Invitrogen, cat#053920).

To formulate the ADCs, DPBS salts in a 20× solution and polysorbate 20(PS20) were added to the protein A elution buffer (Tris-glycine, 50mM-100 mM) at the following final concentrations to stabilize forstorage at 4° C. and during freeze-thaw cycles: CaCl₂ 100 mg/L, MgCl₂(.6H2O) 100 mg/L, KCl 200 mg/L, NaCl 8 g/L, and 0.09 mg/ml PS20. Todetermine protein concentration, for some batches, the OD280 for eachsample was determined on a spectrophotometer. ADC concentration [mg/ml]was calculated by using an extinction coefficient of 1.4 (based on aminoacid sequence). For greater accuracy, ADC concentration was determinedby anti-human IgG Elisa assay (Genway, 40-374-130037).

The first Anti-Her2/neu antibody-based ADCs were produced under theregulation of signal sequences and expression analysis was conducted.ADCs produced were: H1L1 (naked), H2L2 (Phor18-V_(L)-Phor18-V_(H)-IgG1),H2L1 (Phor18-V_(H)-IgG1), H1L2 (Phor18-V_(L)-IgG1), H1L3(C_(L)-Phor18-IgG1) H3L1 (C_(H)3-Phor18-IgG1), H3L3(C_(L)-Phor18-C_(H)3-Phor18-IgG1),H3L4(Phor18-V_(L)-C_(L)-Phor18-C_(H)3-Phor18-IgG1), and H4L3(Phor18-V_(H)-C_(L)-Phor18-C_(H)3-Phor18-IgG1). Based onspectrophotometric (OD₂₈₀) analysis, average yields for ADCs wereH2L2=0.12 mg/L and H1L3=0.8 mg/L

Table 6 shows the individual ADC descriptions, abbreviations, and thenumber and locations of lytic domains (Phor18).

TABLE 6 ADC descriptions and abbreviations ADC Abbreviation Number ofPhor18 lytic Name of ADC (H L) Phor18 location sequences/Antibody IgG1H1 L1 None (‘Naked’) 0 Phor18-V_(L)-IgG1 H1 L2 N-termini light chains(V_(L)) 2 C_(L)-Phor18-IgG1 H1 L3 C-termini light chains (C_(L)) 2Phor18-V_(L)-IgG1-C_(L)-Phor18 H1L 4 N-termini and C-termini lightchains 4 (V_(L), C_(L)) Phor18-V_(L)-Phor18-V_(H)-IgG1 H2 L2 N-terminiheavy and light chains (V_(H), V_(L)) 4 Phor18-V_(H)-IgG1 H2 L1N-termini heavy chains (V_(H)) 2 Phor18-V_(H)-C_(L)-Phor18-IgG1 H2 L3N-termini heavy chains and C-termini 4 light chains (V_(H), C_(L))C_(H)3-Phor18-IgG1 H3 L1 C-termini heavy chain (C_(H)3) 2Phor18-V_(L)-C_(H)3-Phor18-IgG1 H3 L2 N-termini light chains, C-terminiheavy 4 chains (V_(L), C_(H)3) C_(L)-Phor18-C_(H)3-Phor18-IgG1 H3 L3C-termini light and heavy chains (C_(L), 4 C_(H)3)Phor18-V_(L)-C_(L)-Phor18-C_(H)3- H3 L4 N-termini light chains andC-termini both 6 Phor18-IgG1 light and heavy chains (V_(L), C_(L),C_(H)3) Phor18-V_(H)-C_(L)-Phor18-C_(H)3- H4 L3 N-termini heavy chains;C-termini both 6 Phor18-IgG1 heavy and light chains (V_(H), C_(L),C_(H)3) Phor18-V_(H)-C_(H)3-Phor18-IgG1 H4 L1 N- and C-termini heavychains (V_(H), C_(H)3) 4 Phor18-V_(H)-Phor18V_(L)-C_(H)3- H4L2 N-terminiheavy and light chains, C- 6 Phor18-IgG1 termini heavy chains (V_(H),V_(L), C_(H)3)

The quality of ADCs was analyzed on immunoblots of reduced antibodies,allowing heavy and light chains to be visualized. The presence of Phor18was confirmed on the light (L) chain of H1L2 Phor18-V_(L)-IgG1 (H1L2),on the light (L) chain of H1L3 (C_(L)-Phor18-IgG1) and on the heavy (H)chain of H3L1 (C_(H)3-Phor18-IgG1), which has Phor18 only on theC-terminus of the light chain (FIG. 6).

Presence of Phor18 on heavy and light chain was confirmed in westernblotanalysis (FIG. 7A) for the heavy chains with Phor18 conjugation. H1L1and anti-Her2/neu antibody, the naked antibody, served as negativecontrol and did not show a band probing for Phor18. Presence of lightchains in recombinantly produced antibodies and antibody conjugates wereconfirmed in western blot analysis (kappa light chains) for H1L1 (IgG1),H3L1 (C_(H)3-Phor18-IgG1), H1L3 (IgG1-C_(L)-Phor18) and H2L2(Phor18-V_(L)-Phor18-V_(H)-IgG1) had both heavy and light chains basedon IgG western blot analysis, kappa-light chain western blot analysis(FIG. 7B) and anti-Phor18 immuno blots (FIGS. 6 and 7A).

The data indicate that whole antibody-lytic domain (Phor18) conjugatescan be produced recombinantly in a mammalian expression system havingpre-determined stoichiometries of lytic domain (Phor18):AB of 2, 4 and6, and lytic domain (Phor18) in pre-determined locations.

Example 8

This example includes a description of the potency and specificity ofthe eight ADCs.

ADCs with anti-Her-2 receptor (IgG1) conjugated to 2, 4 or 6 Phor18molecules at the N- or C-terminus were analyzed in vitro using aHer2/neu positive ovarian cancer cell line (SKOV-3 human ovarian cancercells) and compared to “naked” antibody. Her-2 receptor negative, ERnegative, PR negative human breast cancer cells (MDA-MB-231) served ascontrol.

In brief, the SKOV-3 (Her2/neu positive, passage number pU51) andMDA-MB-231 (Her2/neu negative, ER negative, PR negative, pu 14) cellswere seeded at a density of 2,000 cells per well in opaque plates inheat inactivated full medium using cell dissociation buffer. After 2days, cells were replenished with fresh media (75 μl) and incubated with25 μl of a 4× serial dilution of each ADC and naked antibody prepared incell culture media were added.

Cells incubated for 4 hours were assayed for membrane integrity using aluminometric assay kit (Promega, Cytotox Glo G9292 lot #317872). Cellviability was determined was determined after 24, 48 and 72 hours usinga luminescent assay kit (Promega, W, Cell Titer Glo, G 7572, lot31511202). Controls for 100% cell viability (culture media) and 100%cell death (0.1% Triton X 100) were incubated under the same conditions.

Data were processed and analyzed to obtain IC₅₀ values using Graph PadPrizm version 5.00 for Windows, GraphPad Software, San Diego Calif. USA,www-graphpad-com (Graph Pad Prizm, Inc). Statistical analysis forsignificance was determined by a two-tailed Student's T-test. Each testwas conducted using double plates of 2-3 wells each to achieve an N of4-6 data points per time point.

The concentration of each ADC was determined spectrophotometrically(OD₂₈₀) and in some cases by IgG determination using ELISA assays. EachADC and naked antibody were prepared from frozen stocks to produce ashighest concentration 800 nM or lower depending on the initialconcentration.

Serial dilutions were prepared in cell culture media to achieve a finalconcentration per well of 0, 0.001, 0.01, 0.1, 1, 10, 100 and 200 nM,corresponding to 0.00015, 0.0015, 0.015, 0.15, 1.5, 15, 30 and 60 μg/mlfor concentrations determined spectrophotometrically. For studies thathad IgG contents the highest possible concentration was tested followedby a 1:1 and 1:10 dilutions for each ADC.

Various time points were included to determine the activity of therecombinantly expressed ADCs. The earliest indication of activity wasmeasured as effect on membrane integration after 4 hours of ADC exposurein Her2/neu positive SKOV-3 cells.

N-terminal conjugated ADC with Phor18 on the variable light chain(Phor18-V_(L)-IgG1) disintegrated the target cell membrane(IC₅₀=187.9±12.8 nM). C-terminal conjugated ADC (C_(H)3-Phor18-IgG1)with one Phor18 molecule had no detectable membrane activity. Nakedanti-Her2-IgG1 also did not detetcably affect membrane integrity.

Table 7 summarizes the results of this activity study. ADCs showed highspecificity for the target cell line SKOV-3 compared to the negativecontrol (Her2/neu negative) MDA-MB-231.

Maximal toxicity levels were determined after 48 hours. The IC₅₀ values[nM] were in the low nanomolar range for N-terminus conjugated ADCPhor18-V_(H)-IgG1 (13.02±2.3 nM, 2 Phor18/AB, N-terminus) and H2L1Phor18-V_(L)-IgG1 (6.9±3.4 nM, 2 Phor18/AB, N-terminus), compared toC-terminus conjugated ADC: C_(H)3-Phor18-IgG1 (27.4±5.1 nM, 2 Phor18/AB,H3L1, C-terminus) having 2 Phor18 molecules per antibody.

N-terminal conjugated ADCs with 4 molecules Phor18 on the N-terminus hadIC₅₀ values of 0.54±0.2 nM (Phor18-V_(L)-Phor18-V_(H)-IgG1, 4 Phor18/AB,N-terminus, H2L2) compared to the C-terminus counterpartC_(L)-Phor18-C_(H)3-Phor18-IgG1 (36.3±10.6 nM, 4 Phor18/AB, H3L3,C-terminus, p<0.001). Lytic domain conjugation at the N-terminus was 70fold more potent than conjugation at the C-terminus.

These data indicate that ADCs having N-terminal Phor18 conjugation weresuperior to C-terminal Phor18 conjugation for stoichiometric ratios of 2and 4 molecules per antibody.

Increase of Phor18 conjugation from 2 to 4 molecules per antibodyresulted in a 12 fold more potent ADC with conjugation at theN-terminus, but not at the C-terminus. ADCs conjugated with 6 Phor18molecules per antibody showed about the same potency with IC50 values of1.1±0.2 nM for Phor18-V_(L)-C_(L)-Phor18-C_(H)3-Phor18-IgG1 (6 Phor18, Nand C terminus, H3L4), and 1.1±0.4 nM forPhor18-V_(H)-C_(L)-Phor18-C_(H)3-Phor18-IgG1 (6 Phor18, H4L3).

Naked anti-Her2-IgG1 showed substantially less cell killing activity inSKOV-3 target cells after 48 hours (IC₅₀ values of 234.6±49 nM). None ofthe ADCs killed target negative MDA-MB-231 cells under the sameconditions.

These data show that lytic domain (Phor18)-conjugated anti-Her-2receptor antibodies (ADCs) are highly specific to Her2/neu expressingcells. Absence of the target receptor leaves cells unharmed.

The potency of lytic domain (Phor18) conjugated antibodies (ADCs) weredependent on the location of conjugation and the number of lytic domain(Phor18) molecules: the highest potency against target cells wereobserved for the N-terminal lytic domain (Phor18)-antibody conjugateswith 2 and 4 lytic domain (Phor18) molecules per antibody. TheN-terminal conjugation exhibited a 70 fold activity increase compared tothe C-terminal lytic domain (Phor18)-antibody conjugation. N-terminalPhor18 conjugated ADCs were more potent compared to C-terminal Phor18conjugates having both 2 and 4 Phor18 molecules per antibody. Inaddition, N-terminal conjugation showed measurable effects on reducingmembrane integrity of the target cells SKOV-3.

Increasing the number of conjugated lytic domain (Phor18) molecules to 6per antibody had maximal activities in the low nanomolar range. Phor18conjugated ADCs were up to 433 fold more potent compared to the nakedantibody.

TABLE 7 In vitro activity of ADCs and naked antibody produced in CHOcells In vitro activity after 48 hours Terminus of IC₅₀ [nM] Conjugationof IC₅₀ [nM] MDA-MB-231 ADC and Naked antibody ADC-ID Phor18 to ABSKOV-3 Her2/neu (+) Her2/neu(−) IgG1 H1L1 None 234.6 ± 49   Not toxicPhor18-V_(L)-IgG1 H1L2 N-termini, L 6.9 ± 3.4 Not toxic chainsPhor18-V_(H)-IgG1 H2L1 N-termini, H 13.02 ± 2.3  Not toxic chainsPhor18-V_(L)-Phor18-V_(H)-IgG1 H2L2 N-termini, H and 0.54 ± 0.2  Nottoxic L chains C_(H)3-Phor18-IgG1 H3L1 C termini H 27.4 ± 5.1  Not toxicchains C_(L)-Phor18-C_(H)3-Phor18-IgG1 H3L3 C-termini H and 36.3 ± 10.6Not toxic L chains Phor18-V_(L)-C_(L)-Phor18-C_(H)3-Phor18-IgG1 H3L4N-termini L 1.1 ± 0.2 Not toxic chains and C chains H chainsPhor18-V_(H)-C_(L)-Phor18-C_(H)3-Phor18-IgG1 H4L3 N-termini H 1.1 ± 0.4Not toxic chains and C- termini L chains

Example 9

This example includes a description of optimization of ADC expressionusing IgG kappa signal sequences. An alternate secretion signal peptidewas evaluated for improvement of quality and expression levels of ADCs.To produce human IgG kappa signal sequence for expression of heavy andlight chains with N-terminal Phor18, new gene synthesis for the L2(Phor18-V_(L)), L4 (Phor18-V_(L)-C_(L)-Phor18), and H2 (Phor18-V_(H))antibody heavy (H) and light (L) chain transcripts with the new signalpeptide at the 5′ end was conducted by Genewiz, Inc. The amino acidsequence of the human IgG kappa signal peptide is MQTDTLLLWVLLLWVPGSTGA(SEQ ID NO.: 154) (Felgenhauer M, et al., Nucleic Acids Res., 18:4927(1990)).

The Phor18-V_(L)-IgG1 and Phor18-V_(L)-Phor18-V_(H)-IgG1 ADCs producedwith the igk secretion signal had a strong signal for light chains onimmunoblots of reduced proteins probed with anti-C_(L) kappa andanti-Phor18. Because the IgG kappa signal peptide induced higherexpression and quality, the igk transcripts were selected for ADCproduction. Production levels of ADCs expressed in CHO cells with theIgG kappa signal sequence, based on Elisa quantitation, were: H1L1=0.8mg/L, C_(L)-Phor18-IgG1=0.8 mg/L, Phor18-V_(L)-IgG1=0.3 mg/L, andPhor18-V_(L)-Phor18-V_(H)-IgG1=0.15 mg/L. Several additional batches ofN-terminal Phor18 ADCs with IgG kappa signal sequence were produced andanalyzed for expression level, quality and efficacy in vitro withcomparable expression levels.

Phor18-V_(L)-IgG1 (H1L2), IgG1-C_(L)-Phor18-IgG1 (H1L3) andPhor18-V_(L)-Phor18-V_(H)-IgG1 (H2L2). These N-terminal and C-terminalconjugated Phor18 ADCs were analyzed for quality and purity byanti-Phor18 immunoblots, IgG and kappa light chain western blot analysis(FIG. 8). Phor18 presence was confirmed on the heavy and light chainsthrough their molecular weights (FIG. 8).

Immunoblot analysis of ADCs showed that the Phor18-V_(L)-IgG1,C_(L)-Phor18-IgG1 and Phor18-V_(L)-Phor18-V_(H)-IgG1 ADCs produced withthe igk secretion signal had heavy and light chains present with Phor18.Expression levels were lower than 0.5 mg/L for both Phor18-V_(L)-IgG1and Phor18-V_(L)-Phor18-V_(H)-IgG1.

Example 10

This example describes studies to characterize the binding kinetics ofHer2/neu protein to Phor18-V_(L)-IgG1 and Phor18-V_(L)-Phor18-V_(H)-IgG1as compared to the binding of Her2/neu protein to Anti-Her2/neuantibody.

Surface plasmon resonance studies were conducted on a BioRad ProteOnsystem using a GLM sensor chip coated with goat anti-human IgG. ADCs(Phor18-V_(L)-IgG1 and Phor18-V_(L)-Phor18-V_(H)-IgG1) and Anti-Her2/neuantibody were diluted to concentrations of 5 μg/ml and injected over theanti-human IgG surface for capture. Anti-Her2/neu antibody was capturedfrom 150 RU to 1000 RU. Phor18-V_(L)-IgG1 andPhor18-V_(L)-Phor18-V_(H)-IgG1 capture levels were between 200 to 900RU.

ErbB2/Her2/neu (Sino Biological #1004-H08H) was analyzed at 50 nM as thehighest concentration in a three-fold dilution series (N=4). Data werecollected at 25 degrees C. Responses from the target surfaces weresubtracted by the reference surface and then globally fit to a 1:1interaction model. Binding constants were determined as shown in Table8.

TABLE 8 Binding constants determined at 25 degrees C. K_(a) (M⁻¹s⁻¹)K_(d) (s⁻¹) K_(D) (pM) Phor18-V_(L)-IgG1 2.20 × 10⁵ 4.43 × 10⁻⁵ 201 ± 24Phor18-V_(L)-Phor18-V_(H)-IgG1 2.05 × 10⁵ 3.55 × 10⁻⁵ 175 ± 13Anti-Her2/neu antibody 3.90 × 10⁵ 3.28 10⁻⁵  84 ± 13

The results indicate that ErbB2 bound to Anti-Her2/neu antibody with anaffinity of 84±13 μM and to Phor18-V_(L)-IgG1 andPhor18-V_(L)-Phor18-V_(H)-IgG1 with affinities that were approximately2.5 and 2-fold weaker at 200±24 and 175±13 pM, respectively. The mostsignificant difference seen was in the association rate forAnti-Her2/neu antibody being about two-fold faster than forPhor18-V_(L)-IgG1 and Phor18-V_(L)-Phor18-V_(H)-IgG1. Thus, the bindingkinetics of Phor18-conjugated to a whole antibody are similar toAnti-Her2/neu antibody, indicating that binding properties are barelyaffected by the conjugation on the variable light and heavy chains.

Example 11

This example describes studies to characterize and evaluate ADCs forcytotoxicity.

Different expression batches were prepared. ADC concentrations weredetermined from spectrophotometric measurements (OC₂₈₀) and in IgGquantification assays. Serial dilutions of selected ADCs with N-terminalPhor18 conjugation for 2 and 4 Phor18 molecules per antibody(Phor18-V_(L)-IgG1 and Phor18-V_(L)-Phor18-V_(H)-IgG1) were prepared asdescribed in Example 10. The in vitro activity was based on IgG contentof each preparation of Phor18-V_(L)-IgG1 andPhor18-V_(L)-Phor18-V_(H)-IgG1.

A 4 h time point was analyzed to determine effects of the ADCs on cellmembrane integrity (Table 8). The ADCs with stoichiometric ratios of 2Phor18/AB and 4 Phor18/AB desintegrated cell membranes of SKOV-3 targetpositive cells after 4 h exposure (IC50 values of 21.5±1 forPhor18-VL-IgG1 and 2.51±0.2 nM for Phor18-V_(L)-Phor18-V_(H)-IgG1),indicating membrane disruption. The membrane activity was 10 fold higherfor N-terminal 4 Phor18 conjugated antibody.

Cell death was measured after 24 hours with IC₅₀ values in the lownanomolar range for Phor18-V_(L)-IgG1 (3.7±0.9 nM) andPhor18-V_(L)-Phor18-V_(H)-IgG1 (0.2±0.04 nM). The maximal potency ofPhor18-V_(L)-I_(G)G1 and Phor18-V_(L)-Phor18-V_(H)-IgG1 was measuredafter 48 hours for Phor18-V_(L)-IgG1 (0.54±0.2 nM) andPhor18-V_(L)-Phor18-V_(H)-IgG1 (0.07±0.02 nM; p<0.0001). In both casesthe in vitro potency for 4 Phor18 antibody conjugates(Phor18-V_(L)-Phor18-V_(H)-IgG1) was 10-20 fold higher than the 2 Phor18antibody conjugate (Phor18-V_(L)-IgG1) (p<0.0001). Naked antibody killedHer2/neu positive target cells (SKOV-3) at 225.8±43 and 295.3±80.6 nMafter 24 and 48 hours.

Consistent with previous results, Phor18-V_(L)-Phor18-V_(H)-IgG1 was themost active ADC showing a 10 fold higher activity thanPhor18-V_(L)-IgG1. The Her2/neu negative control cell line MDA-MB-231was not killed with either naked antibody or ADCs (Phor18-V_(L)-IgG1 orPhor18-V_(L)-Phor18-V_(H)-IgG1) (Table 9). The data demonstrate thathigher numbers of Phor18 (four vs two) conjugated to the N-terminaldomain of the antibody are most potent, compared to antibodies with aC-terminal Phor18 conjugation.

TABLE 9 In vitro activities of recombinantly produced ADCs withN-terminal Phor18 conjugations of 2 and 4 peptides against Her2/neupositive ovarian cancer cell line. In vitro activities Target Cells(SKOV-3) [IC₅₀-nM] 4 hr (Membrane ADC and mAB integrity) 24 hr 48 hrAnti-Her2-IgG1 intact 225.8 ± 43   295.3 ± 80.6 Phor18-V_(L)-IgG1 21.5 ±1.0 3.7 ± 0.9 0.54 ± 0.2 Phor18-V_(L)-Phor18-V_(H)- 2.51 ± 0.2  0.2 ±0.04  0.07 ± 0.02 IgG1

Example 12

This example describes production of ADCs that bind to CD20.

CD 20 is expressed on the surface of B-cell malignancies and representsa surface target that is not internalized. Antibody fragment conjugateswere produced in E. coli (single chain fragments and Phor18-conjugates)and Pichia pastoris (single chain dimers and Phor18-conjugates) andwhole antibody conjugates with 2, 4, and 6 Phor18 molecules wereexpressed in CHO cells. Chemical conjugations with anti-CD20 IgG1antibodies AT80 and MS4A1 were conducted.

Chemical Conjugation—AT80 (Mouse IgG1, Tenovus) and MS4A1 (RituximabLike, R&D)

Purified antibodies IgG1 AT80 and IgG1 MS4A1 were obtained and used forthe chemical conjugation with Phor18. The antibodies were in phosphatebuffered saline (PBS) concentrated to a approximately 2 mg/mL. A 20 mMsolution of SPDP was freshly prepared in DMSO, and added to the antibodysolution in 20-fold excess. The mixture was incubated at roomtemperature for about 30 minutes to produce the antibody-linkerintermediate. Excess unreacted SPDP is removed by size exclusionchromatography. The cytotoxin molecule containing cysteine wasthoroughly reduced by reaction with a 10-fold excess of reductacrylreagent before mixing in 10-fold excess with the antibody-linkerconstruct. The reaction is allowed to incubate at room temperature for18 hours, then desalted to remove unreacted cytotoxin molecule. Thesolution is filter-sterilized before storage. Concentrations of finalADCs were for the AT80-Phor18 conjugate 0.76 mg/ml and for theMS4A1-Phor18 conjugate 0.34 mg/ml as determined by Bradford proteinmeasurements. Typical number of Phor18 molecules per antibody using theSPDP method for conjugation is 3-5 molecules of Phor18 per antibody.

Two antibodies against CD20 chemically conjugated to Phor18 wereanalyzed because these bind to different domains of the extracellularCD20 loops. To compare, in in vitro studies, the cytotoxicity of twochemically conjugated CD20 targeting whole antibody IgG1-Phor18conjugates with “naked” antibody (IgG1) in CD20 positive cells (Daudiand Raji, Burkitt's lymphoma). CD20 negative leukemia cells (U937)served as controls.

Naked antibody anti-CD20-IgG1-Phor18 were chemically conjugated (MS4Aand AT80), Mw app.158,000 g/mol at a concentrations of 0.34 mg/ml(MS4A-Phor18) and 0.76 mg/ml (AT-80-Phor18). Cell lines were obtained atthe American Type Cell Collection (Mannassas, Va.). Human Non-Hodgkin'slymphoma cells Daudi (CD20 positive, passage number p2), Raji (CD20positive p 2) and human leukemia cell line U937 (CD20 negative, p 10)were seeded at a density of 3,000 cells per well in opaque plates inheat inactivated full medium. After 24 hours cells were replenished withfresh media (75 μl) and incubated with 25 μl of a 4× serial dilution ofMS4A1-Phor18 and AT80-Phor18 of 0.001, 0.01, 0.1, 1, 10, 100 and 500 nM(N=6). Cells incubated for 2-5 hours were assayed for membrane integrityusing a luminometric assay kit (Promega, Madison, Wis., Cytotox GloG9292 lot #301329). Cell viability was determined was determined after24, and 48 hours using a luminescent assay kit (Promega, Madison, Wis.,Cell Titer Glo, G 7572, lot 30068102).

Chemically conjugated MS4A1-Phor18 and AT80-Phor18 were tested for theirmembrane activity. MS4A1-Phor18 was not active after 2 or 5 hours inCD20 positive cell lines Raji and Daudi, whereas AT-80-Phor18 destroyedmembrane integrity in CD20 positive Daudi cells with a IC₅₀ value of106.1±2.9 nM. Daudi cells were killed by the AT-80-Phor18 conjugatewithin 48 h with IC₅₀ values of 11.9±0.9 nM and Raji cells with IC₅₀values of 6.3±1.02 nM. The CD20 negative control cell line U937 was notkilled by either naked AT-80 antibody or AT-80-Phor18 conjugates.

The MS4A1-Phor18 conjugate showed low activity in Raji cells with IC₅₀values of 267±13 and 227±11 nM after 24 and 46 hours. Daudi cells weremuch more sensitive to the MS4A1-Phor18 conjugate with IC₅₀ values of10.6±2.1 and 3.0±1.2 nM. The naked MS4A1 antibody was not toxic toeither Raji or Daudi cell lines. The CD20 negative human leukemia cells(U937) were not killed by either of the ADCs (Table 10).

These data show that chemically conjugated Phor18 ADCs kill CD20positive cells on contact, and do not appear to require internalization.Cytotoxicity is specific for CD20 target and depends on the bindingdomain of the ADC.

TABLE 10 In vitro activities of chemically conjugated anti-CD20IgG1-Phor18 conjugates in CD20 positive Non-Hodgkin's lymphoma celllines Raji and Daudi and the CD20 negative cell line U937 Anti-CD20-Anti-CD20-Phor18 Phor18 (AT80) Anti-CD20 AT80 (MS4A1) Anti-CD20 MS4A1[IC₅₀ nM] [IC₅₀ nM] [IC₅₀ nM] [IC₅₀ nM] Raji 2 h 120.8 ± 0.45 Not toxicND ND 5 h (N = 6) 138.2 ± 28.8 Not toxic ND ND 24 h ND ND 267.1 ± 13  Not toxic 46 h  6.3 ± 1.02 ND 227 ± 11 Not toxic Daudi 2 h 126.5 ± 12.1Not toxic Not toxic Not toxic 5 h (N = 6) 106.1 ± 2.9  Not toxic Nottoxic Not toxic 24 h ND ND 10.6 ± 2.1 Not toxic 46 h 11.9 ± 0.9 115.5 ±53  3.0 ± 1.2 Not toxic U937 (CD20 2-46 h Not toxic Not toxic Not toxicNot toxic ND = not determined

Example 13

This example describes a comparison of caspase activation of CD20targeting ADCs.

CD20 targeting ADCs are not internalized. Initiation of cell death canbe measured by determining apoptosis related pathways. Early apoptosisprocesses shows activation of caspases 3 and 7. Caspases are members ofthe cysteine aspartic acid-specific protease family and play keyeffector role in apoptosis in mammalian cells. The assay provides aluminogenic caspase-3/7 substrate, which contains the tetrapeptidesequence DEVD, in a reagent optimized for caspase activity, luciferaseactivity and cell lysis.

To compare, in in vitro studies, the caspase activation of chemicallyconjugated CD20 targeting whole antibody IgG1-Phor18 conjugate with“naked” antibody (IgG1-AT80) in CD20 positive cells (Daudi Burkitt'slymphoma).

Human Non-Hodgkin's lymphoma cells Daudi (CD20 positive, passage numberp2), were seeded at a density of 3,000 cells per well in opaque platesin heat inactivated full medium. After 24 hours cells were fed withfresh media (75 μl) and incubated with AT80-Phor18 (0.76 mg/ml) or nakedantibody AT-80 of 15 and 75 μg/ml (100 and 500 nM) (N=6). Staurosporineat 10 μM served as positive control for caspase 3/7 activation. After 5hours of incubation cultures were assayed for caspase 3/7 levels using aluminometric assay kit (Promega, Madison, Wis., Caspase Glo 3/7 G811Clot #28731802).

Caspase 3/7 activation was calculated from relative light units fromluminometric signals. Staurosporine was set at 100% caspase 3/7activation, cell suspension alone without additions of reagents servedas 0% caspase levels. AT80-Phor18 elevated caspase 3/7 levels to 13±2.6%at 15 μg/ml and reached 85.7±5 at 75 μg/ml. Unconjugated AT-80 at 15 or75 μg/ml concentrations lacked caspase activation (−12.3 and −2.3%). Thehighest dose of AT80-Phor18 resulted in a caspase activation that wascomparable to Staurosporine (p=0.06) (FIG. 9).

Phor18-conjugated ADCs activate caspase 3/7 when bound to the targetcells thus promoting apoptotic cell death within 5 hours.

Example 14

This example describes expression constructs and characterization ofsingle chain anti-CD20 Phor18 (scFv-Phor18) conjugates in E. coli.

A single chain Fv anti-CD20 fragment was designed by inserting RituxanCDRs into the humanized variable regions of p185. A poly-histidine tagwas added to the N-terminus of the protein for purification. The aminoacid sequence is shown below with the inserted CDRs bolded. Apoly-glycine flexible linker was inserted between the V_(L) and V_(H)domains and Phor18 was placed at the C-terminus after a GS linker.

Amino acid sequence of the scFv fragment (naked AB); CDRs in bold (SEQ ID NO.: 92)        10         20         30         40         50         60HHHHHHDIQL TQSPAILSAS PGEKVTMTCR ASSSVSYIHW FQQKPGSSPK PWIYATSNLA        70         80         90        100        110        120SGVPVRFSGS GSGTSYSLTI SRVEAEDAAT YYCQQWTSNP PTFGGGTKLE IGSTSGGGSG       130        140        150        160        170        180GGSGGGGSSV QLQQPGAELV KPGASVKMSC KASGYTFTSY NMHWVKQTPG RGLEWIGAIY       190        200        210        220        230        240PGNGDTSYNQ KFKGKATLTA DKSSSTAYMQ LSSLTSEDSA VYYCARSTYY GGDWYFDVWGQGTTVTVSS Amino acid sequence of the scFv-Phor18 conjugate (C-terminus);CDRs in bold  (SEQ ID NO.: 93)        10         20         30         40         50         60HHHHHHDIQL TQSPAILSAS PGEKVTMTCR ASSSVSYIHW FQQKPGSSPK PWIYATSNLA        70         80         90        100        110        120SGVPVRFSGS GSGTSYSLTI SRVEAEDAAT YYCQQWTSNP PTFGGGTKLE IGSTSGGGSG       130        140        150        160        170        180GGSGGGGSSV QLQQPGAELV KPGASVKMSC KASGYTFTSY NMHWVKQTPG RGLEWIGAIY       190        200        210        220        230        240PGNGDTSYNQ KFKGKATLTA DKSSSTAYMQ LSSLTSEDSA VYYCARSTYY GGDWYFDVWG       250        260 QGTTVTVSSG SKFAKFAKKF AKFAKKFAK

This antibody fragment was ordered from Genscript USA (Piscataway, N.J.)for production in E. coli. Genscript used their pGS21a expressionplasmid for production in E. coli Arctic Express cells. Genscriptisolated the protein from E. coli inclusion bodies.

The naked scFv fragment had was purified through affinity chromatographyand resulted in a yield of 15 mg/L at a concentration of 0.31 mg/ml. thepurity was determined as 85%. The molecular weight was determined usingCoomassie stained SDS PAGE analysis with 27,088 g/mol. The Phor18conjugate had a similar yield of 15 mg/L and a purity of 85% based onCoomassie stained SDS-Page. The molecular weight was measured at 29,349g/mol. Both naked AB and ADC were provided in 50 mM Tris buffer, pH 8.0.

Anti-CD20 ScFv-Phor18 has a calculated molecular weight if 29.3 kD. Theantibody fragment was expressed with a poly-histidine tag forpurification. An immunoblot of the protein probed with anti-histidineand SDS-PAGE stained with coomassie blue indicated the molecular weightwas in the correct range.

Example 15

This example describes expression and characterization of recombinantlyproduced CD20 targeting Fv-C_(H)3-C_(H)3-Fv-Phor18 conjugates in E.coli:

A bivalent anti-CD20 minibody was designed by adding a flexibleglycine-serine linker between the variable domains and between theC_(H)3 domains to result in a FV-C_(H)3-C_(H)3-F minibody. Apoly-histidine tag was added to the N-terminus of the protein forpurification from inclusion bodies and Phor18 was placed at theC-terminus after a GS linker. The amino acid sequences are shown belowwith the inserted CDRs bolded in variable domains and the C_(H)3 domainare in lower case letters.

Amino acid sequence of the anti-CD20 minibody Fv-C_(H)3-C_(H)3-Fv;CDRs in bold  (SEQ ID NO. 94)        10         20         30         40         50         60DIQLTQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR        70         80         90        100        110        120FSGSGSGTSY SLTISRVEAE DAATYYCQQW TSNPPTFGGG TKLEIGSTSG GGSGGGSGGG       130        140        150        160        170         180GSSVQLQQPG AELVKPGASV KMSCKASGYT FTSYNMHWVK QTPGRGLEWI GAIYPGNGDT       190        200        210        220        230        240SYNQKFKGKA TLTADKSSST AYMQLSSLTS EDSAVYYCAR STYYGGDWYF DVWGQGTTVT       250        260        270        280        290        300VSSGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV       310        320        330        340        350        360LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK GSTSGGGSGG       370        380        390        400        410        420GSGGGGSSGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK       430        440        450        460        470        480TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKVQLQQ       490        500        510        520        530        540PGAELVKPGA SVKMSCKASG YTFTSYNMHW VKQTPGRGLE WIGAIYPGNG DTSYNQKFKG       550        560        570        580        590        600KATLTADKSS STAYMQLSSL TSEDSAVYYC ARSTYYGGDW YFDVWGQGTT VTVSSGSTSG       610        620        630        640        650        660GGSGGGSGGG GSSDIQLTQS PAILSASPGE KVTMTCRASS SVSYIHWFQQ KPGSSPKPWI       670        680        690        700        710YATSNLASGV PVFSGSGSGT SYSLTISRVE AEDAATYYCQ QWTSNPPTFG GGTKLEIAmino acid sequence of the anti-CD2O-Phor18 minibody conjugateFv-C_(H)3-C_(H)3-Fv-Phor18; CDRs in bold (SEQ ID NO.: 95)        10         20         30         40         50         60DIQLTQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR        70         80         90        100        110        120FSGSGSGTSY SLTISRVEAE DAATYYCQQW TSNPPTFGGG TKLEIGSTSG GGSGGGSGGG       130        140        150        160        170         180GSSVQLQQPG AELVKPGASV KMSCKASGYT FTSYNMHWVK QTPGRGLEWI GAIYPGNGDT       190        200        210        220        230        240SYNQKFKGKA TLTADKSSST AYMQLSSLTS EDSAVYYCAR STYYGGDWYF DVWGQGTTVT       250        260        270        280        290        300VSSGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV       310        320        330        340        350        360LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK GSTSGGGSGG       370        380        390        400        410        420GSGGGGSSGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK       430        440        450        460        470        480TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKVQLQQ       490        500        510        520        530        540PGAELVKPGA SVKMSCKASG YTFTSYNMHW VKQTPGRGLE WIGAIYPGNG DTSYNQKFKG       550        560        570        580        590        600KATLTADKSS STAYMQLSSL TSEDSAVYYC ARSTYYGGDW YFDVWGQGTT VTVSSGSTSG       610        620        630        640        650        660GGSGGGSGGG GSSDIQLTQS PAILSASPGE KVTMTCRASS SVSYIHWFQQ KPGSSPKPWI       670        680        690        700        710YATSNLASGV PVFSGSGSGT SYSLTISRVE AEDAATYYCQ QWTSNPPTFG GGTKLEIGSK       730 FAKFAKKFAK FAKKFAK

The antibody fragment was obtained from Genscript USA (Piscataway, N.J.)for production in E. coli. Genscript used their pGS21a expressionplasmid for production in E. coli Arctic Express cells. Genscriptisolated the protein from E. coli inclusion bodies.

The naked anti-CD20 minibody had was purified through affinitychromatography and resulted in a yield of 3 mg/L at a concentration of0.23 mg/ml. the purity was determined as 80%. The molecular weight wasdetermined using Coomassie stained SDS PAGE analysis with 78,988 g/mol.The Phor18 conjugate had a similar yield of 3 mg/L and a purity of 70%based on Coomassie stained SDS-Page. The molecular weight was measuredat 93,515 g/mol. Both naked AB and ADC were provided in 50 mM Trisbuffer, 150 mM NaCl, 15-20% glycerol, pH 8-9.5.

The naked single chain Fv and anti-CD20 minibody and scFv-Phor18 andanti CD20-Phor18 conjugates were expressed in E. coli. Distinctmolecular weights that corresponded to each antibody fragment backbonewere demonstrated on SDS-PAGE gels and Western blots.

Example 16

This example describes the in vitro analysis of the scFv and anti-CD20naked minibody and Phor18 conjugates in CD20 positive and CD20 negativecell lines.

To compare, in in vitro studies, the cytotoxicity of a recombinantlyproduced CD20 targeting scFv-Phor18 and anti-CD20-Phor18 minibody in abacterial expression system with “naked” antibody (scFv and anti-CD20minibody) in CD20 positive cells (Daudi, Burkitt's lymphoma). CD20negative leukemia cells (U937) served as controls.

The concentration of each naked antibody fragment and ADC was determinedaccording to Bradford. Naked antibody scFv Mw 27,088 g/mol at aconcentration of 0.3 mg/ml, minibody Mw 78,988 g/mol, 0.231 mg/ml; Phor18 conjugated antibodies were scFv-Phor18 (Mw 29,349 g/mol, 0.9 mg/ml)and minibody-Phor18 (Mw 93,515, 0.48 mg/ml). Cell lines were obtained atthe American Type Cell Collection (Mannassas, Va.). Human Non-Hodgkin'slymphoma cells Daudi (CD20 positive, passage number p8) and humanleukemia cell line U937 (CD20 negative, p 16) were seeded at a densityof 2,000 cells per well in opaque plates in heat inactivated fullmedium. After 24 hours cells were replenished with fresh media (75 μl)and incubated with 25 μl of a 4× serial dilution of scFv-Phor18 andminibody-Phor18 ADC and naked antibody fragment (scFv and minibody)prepared in cell culture media were added at concentrations of 0.0001,0.001, 0.01, 0.1, 1, 10, 100, 200, and 500 nM (N=6). Cells incubated for4 hours were assayed for membrane integrity using a luminometric assaykit (Promega, Madison, Wis., Cytotox Glo G9292 lot #301329). Cellviability was determined was determined after 24, 48 and 72 hours usinga luminescent assay kit (Promega, Madison, Wis., Cell Titer Glo, G 7572,lot 30062102).

Controls for 100% cell viability (culture media) and 100% cell death(0.1% Triton X 100) incubated under the same conditions.

Data were processed and analyzed to obtain IC₅₀ values using Graph PadPrizm version 5.00 for Windows, GraphPad Software, San Diego Calif. USA,www-graphpad-com (Graph Pad Prizm, Inc). Statistical analysis forsignificance was determined by a two-tailed Student's T-test. Each testwas conducted using 2 plates with 2-3 wells each to achieve an N of 4-6data points per time point.

Recombinant scFv-Phor18 conjugates were expressed in E. coli. As shownin FIG. 10, Table 11, the anti-CD20-scFv-Phor18 conjugate destroyedmembrane integrity in CD20 positive Daudi cells. Human Burkitts lymphomacells (Daudi) were killed within 48 h, whereas the CD20 negative humanleukemia cells (U937) were not killed. Naked scFv antibody did not killany of the cell lines. Hill plot analysis of the cell viability dataresulted in IC₅₀ values for the minibody conjugate of 10.02±0.5 nM after24 h and 1.5±0.3 nM after 48 h in Daudi cells. Naked scFv was not toxic.The CD20 negative cell line U937 was not killed by either naked scFv orscFv-Phor18 or minibody-Pho18 conjugates. In vitro activities of theminibody-Phor18 conjugates were 10.5±0.5 and 3.9±1.6 nM in CD20 positiveDaudi cells after 24 and 46 hours. Naked minibody was not toxic.

TABLE 11 In vitro toxicities of scFv-Phor18 andFv-C_(H)3-C_(H)3-Fv-Phor18, and scFv and Fv-C_(H)3-C_(H)3-Fv targetingCD 20. Daudi cells (NHL) are positive for CD 20, U937 cells (leukemia)are negative for CD20. Fv-C_(H)3- Fv-C_(H)3-C_(H)3-Fv- scFv scFv-Phor18C_(H)3-Fv Phor18 [IC₅₀ nM] [IC₅₀ nM] [IC₅₀ nM] [IC₅₀ nM] Daudi 2 h Nottoxic 109.8 ± 0.9 ND ND 5 h (N = 8) Not toxic  111.6 ± 15.1 ND ND 24 h(N = 6) Not toxic 10.02 ± 0.5 Not toxic 10.5 ± 0.5 46 h (N = 6) Nottoxic  1.5 ± 0.3 Not toxic  3.6 ± 1.6 U937 2-48 h Not toxic Not toxicNot toxic Not toxic ND = Not Determined

Potent scFv-Phor18 and Fv-C_(H)3-C_(H)3-Fv-Phor18 conjugates wereexpressed in E. coli and were more potent than naked scFv orFv-C_(H)3-C_(H)3-Fv. CD20 targeted ADCs killed specifically targetcells—cell death was independent on internalization. scFv-Phor18 orFv-C_(H)3-C_(H)3-Fv-Phor18 targeted conjugates but not the naked scFvantibody fragment activated apoptotic pathways. C-terminus conjugatedscFv and Fv-C_(H)3-C_(H)3-Fv conjugates showed similar activities in thenanomolar range after 24 hours.

Example 17

This example describes a possible mechanism of action of the CD20targeted ADC.

In in vitro studies, caspase activation of recombinantly expressed CD20targeting scFv-Phor18 conjugate with “naked” antibody (scFv) in CD20positive cells (Daudi Burkitt's lymphoma) was compared. HumanNon-Hodgkin's lymphoma cells Daudi (CD20 positive, p 5) and Raji (CD20positive, passage number p 3), were seeded at a density of 3,000 cellsper well in opaque plates in heat inactivated full medium. After 24hours cells were fed with fresh media (75 μl) and incubated withscFv-Phor18 (0.365 mg/ml) or naked antibody scFv of 5 and 15 μg/ml (100and 500 nM) (N=6). Staurosporine at 10 μM served as positive control forcaspase 3/7 activation. After 5 hours were assayed for caspase 3/7activation using a luminometric assay kit (Promega, Madison, Wis.,Caspase Glo 3/7 G811C lot #28731802).

Relative caspase 3/7 activation was calculated from relative light unitsfrom luminometric signals. Staurosporine was set at 100% caspase 3/7activation and no reagents for 0% activation controls. scFv-Phor18 at 5μg/ml had elevated caspase 3/7 levels of 18.3±0.8 and 29.3±0.9% comparedto Staurosporine (FIG. 11).

Phor18-conjugated ADCs activate caspase 3/7 in target cells thuspromoting apoptotic cell death. Accordingly, antibody Phor18 conjugatesas single chain or minibody fragments can be produced recombinantly andare active without internalization on target cells. Caspase activationthrough ADCs may be a possible mechanism of action.

Example 18

This example describes the expression of CD20 targeted scFvFc Phor18conjugates having Phor18 conjugated at the N or C terminus and having astoichiometry of 1 and 2 Phor18 molecules per antibody fragment.

The recombinant series was produced by Genscript, USA (Piscataway, N.J.)in E. coli using periplasmatic secretion. Transcripts were synthesizedand codon usage was optimized for E. Coli The Genscript pGS-21aexpression vector was used. The expressed proteins were directed to theperiplasm of E. Coli cells by addition of a cleavable PelB bacterialsignal sequence, composed of the amino acids MKYLLPTAAAGLLLLAAQPAMA (SEQID NO.:101), to the N-terminus. The Rituxan CDRs were inserted into thehumanized variable regions of p185 Herceptin and are shown in bold. Fourglycines and a serine linked the variable regions for added flexibility.Phor18 was positioned at the N-terminus, the C-terminus, or both. Theamino acid sequence of the antibody fragment are as follows:

Cleaved PelB N-terminal signal sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ IDNO.:96)

Amino acid sequences of scFv-Fc (naked antibody); CDRs in bold (SEQ ID NO. 97)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG KAPKLLIYAT SNLASGVPSR        70         80         90        100        110        120FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG TKVEIKGGGG SEVQLVESGG       130        140        150        160        170         180GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA AIYPGNGDTS YNQKFKGRFT       190        200        210        220        230        240ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD VWGQGTLVTV SSVQPCPAPE       250        260        270        280        290        300LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VENAKTKPRE       310        320        330        340        350        360EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP       370        380        390        400        410        420SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD       430        440        450 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKAmino acid sequences of lytic-peptide Phor18-antibody conjugate;CDRs in bold Phor18-scFv-Fc-Phor18: N- and C-terminal conjugation(SEQ ID NO.: 98)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG        70         80         90        100        110        120KAPKLLIYAT SNLASGVPSR FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG       130        140        150        160        170         180TKVEIKGGGG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA       190        200        210        220        230        240AIYPGNGDTS YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD       250        260        270        280        290        300VWGQGTLVTV SSVQPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV       310        320        330        340        350        360KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE       370        380        390        400        410        420KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT       430        440        450        460        470        480TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKGSKFAK       490 FAKKFAKFAK KFAKAmino acid sequences of lytic-peptide Phor18-antibody conjugate;CDRs in bold Phor18-scFv-Fc: N-terminal conjugation (SEQ ID NO.: 99)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG        70         80         90        100        110        120KAPKLLIYAT SNLASGVPSR FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG       130        140        150        160        170         180TKVEIKGGGG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA       190        200        210        220        230        240AIYPGNGDTS YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD       250        260        270        280        290        300VWGQGTLVTV SSVQPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV       310        320        330        340        350        360KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE       370        380        390        400        410        420KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT       430        440        450        460        470 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKAmino acid sequences of lytic-peptide Phor18-antibody conjugate;CDRs in bold Phor18-scFv-Fc: C-terminal conjugation (SEQ ID NO.: 100)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG KAPKLLIYAT SNLASGVPSR        70         80         90        100        110        120FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG TKVEIKGGGG SEVQLVESGG       130        140        150        160        170         180GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA AIYPGNGDTS YNQKFKGRFT       190        200        210        220        230        240ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD VWGQGTLVTV SSVQPCPAPE       250        260        270        280        290        300LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VENAKTKPRE       310        320        330        340        350        360EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP       370        380        390        400        410        420SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD       430        440        450        460        470 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKGSKFAK FAKKFAKFAK KFAK

The ADCs were purified over protein A affinity chromatography columnsand stored frozen (−20 degrees C.). The concentration of each nakedantibody fragment and ADC was determined spectrophotometrically (OD₂₈₀).Naked antibody scFv-Fc Mw 52,562 g/mol at a concentration of 1.1 mg/ml,Phor 18 conjugated antibodies were Phor18-V_(L)-scFvFc (Mw 54,685 g/mol,1.0 mg/ml), Phor18-C_(H)3-scFvFc (Mw 54,685 g/mol, 0.8 mg/ml) formedsingle chains.

Example 19

This example describes activity of scFv-Fc-Phor18 conjugates in in vitrostudies.

The cytotoxicity of recombinantly produced CD20 targeting scFv-Fc-Phor18conjugates in a E. coli expression system with Phor18 conjugations at N-or C-terminus and at N- and C-terminus was compared to “naked” antibody(scFv-Fc) in CD20 positive cells (Daudi, Burkitts lymphoma). CD20negative leukemia cells (U937) served as controls. E. coli expressedADCs represented single chains and were conjugated to 1 Phor18 moleculeat the N-terminus of the V_(L) chain (Phor18-V_(L)-scFv-Fc), at theC-terminus of the C_(H)3 chain (scFv-Fc-C_(H)3-Phor18).

The concentration of each naked antibody fragment and ADC was determinedspectrophotometrically (OD₂₈₀). Naked antibody scFv-Fc Mw 52,562 g/molat a concentration of 1.1 mg/ml, Phor18 conjugated antibodies werePhor18-V_(L)-scFvFc (Mw 54,685 g/mol, 1.0 mg/ml), scFv-Fc-C_(H)3-Phor18(Mw 54,685 g/mol, 0.8 mg/ml). Human Non-Hodgkin's lymphoma cells Daudi(CD20 positive, passage number p7) and human leukemia cell line U937(CD20 negative, p 6) were seeded at a density of 2,000 cells per well inopaque plates in heat inactivated full medium using cell dissociationbuffer. After 24 hours cells were replenished with fresh media (75 μl)and incubated and incubated with 25 μl of a 4× serial dilution of eachADC and naked antibody prepared in cell culture media were added atconcentrations of 0.01, 0.1, 1, 10, 100, 200 and 500 nM for scFvFc,Phor18-V_(L)-scFvFc and scFv-Fc-C_(H)3-Phor18.

Cells incubated for 4 hours were assayed for membrane integrity using aluminometric assay kit (Promega, Madison, Wis., Cytotox Glo G9292 lot#26229601). Cell viability was determined was determined after 24 hoursusing a luminescent assay kit (Promega, Madison, Wis., Cell Titer Glo, G7572, lot 30731602). Controls for 100% cell viability (culture media)and 100% cell death (0.1% Triton X 100) incubated under the sameconditions.

Data were processed and analyzed to obtain IC50 values using Graph PadPrizm version 5.00 for Windows, GraphPad Software, San Diego Calif. USA,www-graphpad-com (Graph Pad Prizm, Inc). Statistical analysis forsignificance was determined by a two-tailed Student's T-test. Each testwas conducted using 2 plates with 2-3 wells each to achieve an N of 4-6data points per time point.

Recombinant ScFv-Fc-Phor18 conjugates with 1 Phor18 on the N-terminus orthe C-terminus were expressed in E. coli. As shown in Table 12, theanti-CD20-Phor18 conjugate Phor18-V_(L)-scFvFc destroyed membraneintegrity in CD20 positive Daudi cells after 4 h with IC₅₀ values of277±37.5 nM. A tenfold higher value was obtained for the C-terminalconjugate for scFv-Fc-C_(H)3-Phor18 with 2558±259 nM. Naked scFvFc wasnot toxic.

Human Burkitts lymphoma cells (Daudi) were killed within 24 h with IC₅₀values of 21.8±0.8 nM for Phor18-V_(L)-scFv-Fc, and 422.6±47.5 nM forscFv-Fc-C_(H)3-Phor18.

Naked scFv caused cell killing at 677.2±45.3 nM in the CD20 positiveDaudi cell line. The CD20 negative human leukemia cells (U937) was notkilled after 4 hours and showed compared to the target cell line lowersensitivity with IC₅₀ values of 495.4±35.2 nM for the naked scFvFc,105.3±15.6 nM Phor18-V_(L)-scFv-Fc and 722.3±33.2 nM for thescFv-Fc-C_(H)3-Phor18 conjugates.

TABLE 12 In vitro toxicities of Phor18-V_(L)-scFv-Fc andscFv-Fc-C_(H)3-Phor18, and scFv-Fc targeting CD 20. Daudi cells (NHL)are positive for CD 20, U937 cells (leukemia) are negative for CD20.scFvFc Phor18-V_(L)-scFv-Fc scFv-Fc-C_(H)3-Phor18 naked AB IC₅₀ values[nM] IC₅₀ values [nM] Daudi 4 h (N = 8) Not toxic   277 ± 37.5 2558 ±259 24 h (N = 8) 677.2 ± 45.3 21.8 ± 0.8 422.6 ± 47.5 U937 4 h (N = 8)Not toxic Not toxic Not toxic 24 h (N = 8) 495.4 ± 35.2 105.3 ± 15.6722.3 ± 33.2

N-terminus conjugated Phor18 antibody fragments were more toxic comparedto C-terminus conjugated Phor18 antibody fragments.

Example 20

This example describes expression and activity of CD20 targetingscFv-Fc-Phor18 conjugates produced in Pichia Pastoris (yeast).

Four anti-CD20 single chain Fv-Fc antibody fragments were designed byinserting the Rituxan CDRs into the humanized variable regions of p185.The inserted CDRs are bolded in the amino acid sequences shown below. Apoly-G linker was used between the variable regions and human constantdomains hinge, C_(H)2 and C_(H)3. The order of the antibody fragmentsequences shown below are as follows: naked antibody, C-terminal andN-terminal Phor18, N-terminal Phor18 only, C-terminal Phor18 only. Theamino acid sequences for each construct are shown below:

Amino acid sequences of naked antibody; CDRs in bold (SEQ ID NO.: 101)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG KAPKLLIYAT SNLASGVPSR        70         80         90        100        110        120FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG TKVEIKGGGG SGGGGSGGGG       130        140        150        160        170        180SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA AIYPGNGDTS       190        200        210        220        230        240YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD VWGQGTLVTV       250        260        270        280        290        300SSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD       310        320        330        340        350        360GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK       370        380        390        400        410        420GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS       430        440        450        460        470 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKAmino acid sequences of conjugated antibody Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18;CDRs in bold (SEQ ID NO.: 102)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG        70         80         90        100        110        120KAPKLLIYAT SNLASGVPSR FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG       130        140        150        160        170        180TKVEIKGGGG SGGGGSGGGG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ       190        200        210        220        230        240APGKGLEWVA AIYPGNGDTS YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS       250        260        270        280        290        300TYYGGDWYFD VWGQGTLVTV SSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT       310        320        330        340        350        360CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK       370        380        390        400        410        420CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE       430        440        450        460        470        480WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS       490        500 LSLSPGKGSK AFKKAFKAFK KAFKAFKAmino acid sequences of conjugated antibody Phor18-V_(L)-scFv-Fc;CDRs in bold  (SEQ ID NO.: 103)        10         20         30         40         50         60KFAKFAKKFA KFAKKFAKGS DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG        70         80         90        100        110        120KAPKLLIYAT SNLASGVPSR FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG       130        140        150        160        170        180TKVEIKGGGG SGGGGSGGGG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ       190        200        210        220        230        240APGKGLEWVA AIYPGNGDTS YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS       250        260        270        280        290        300TYYGGDWYFD VWGQGTLVTV SSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT       310        320        330        340        350        360CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK       370        380        390        400        410        420CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE       430        440        450        460        470        480WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKSLSLSPGKAmino acid sequences of conjugated antibody scFv-Fc-C_(H)3-Phor18;CDRs in bold (SEQ ID NO.: 104)        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG KAPKLLIYAT SNLASGVPSR        70         80         90        100        110        120FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG TKVEIKGGGG SGGGGSGGGG       130        140        150        160        170        180SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA AIYPGNGDTS       190        200        210        220        230        240YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD VWGQGTLVTV       250        260        270        280        290        300SSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD       310        320        330        340        350        360GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK       370        380        390        400        410        420GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS       430        440        450        460        470        480DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGSK AFKKAFKAFKKAFKAFKThe gene synthesis for the four scFv-Fc fragments was ordered fromGenescript USA and codons were optimized for production in PichiaPastoris yeast strain GS 115 at Genscript. Genescript subcloned eachexpression plasmid into the InVitrogen yeast expression plasmid pPICZαA(cat#V195-20 lot#900479, FIG. 12). This expression plasmid has a yeastα-factor secretory signal so that the antibody protein could be isolatedfrom medium.

Characterization of CD20 targeting scFv-Fc-Phor18 conjugates on silverstained SDS PAGE showed that Pichia pastoris expressed ADCs representedsingle chain dimers and were conjugated to 2 or 4 Phor18 molecules atthe N-terminus of the V_(L) chain (Phor18-V_(L)-scFvFc), at theC-terminus of the C_(H)3 chain (scFvFc-C_(H)3-Phor18) and at theN-terminus of the V_(L) chains and the C-terminus of the C_(H)3 chain(Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18).

The concentration of each naked antibody fragment and ADC was determinedspectrophotometrically (OD₂₈₀). Naked antibody scFv-Fc (Mw 102,078g/mol) at a concentration of 0.26 mg/ml, Phor18 conjugated antibodieswere Phor18-V_(L)-scFvFc (Mw 106,616 g/mol, 0.5 mg/ml),scFvFc-C_(H)3-Phor18 (Mw 106,616 g/mol, 0.17 mg/ml) andPhor18-V_(L)-scFv-Fc-C_(H)3-Phor18 (Mw 111,154 g/mol, 0.7 mg/ml).

To compare, in in vitro studies, the cytotoxicity of recombinantlyproduced CD20 targeting scFv-Fc-Phor18 conjugates in a yeast expressionsystem with “naked” antibody (scFv-Fc) in CD20 positive cells (Daudi,Burkitts lymphoma). CD20 negative leukemia cells (U937) served ascontrols. Pichia expressed ADCs represented single chain dimers and wereconjugated to 2 or 4 Phor18 molecules at the N-terminus of the V_(L)chain (Phor18-V_(L)-scFvFc), at the C-terminus of the C_(H)3 chain(scFvFc-C_(H)3-Phor18) and at the N-terminus of the V_(L) chains and theC-terminus of the C_(H)3 chain (Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18).

Human Non-Hodgkin's lymphoma cells Daudi (CD20 positive, passage numberp3) and human leukemia cell line U937 (CD20 negative, p 12) were seededat a density of 2,000 cells per well in opaque plates in heatinactivated full medium using cell dissociation buffer. After 24 hourscells were replenished with fresh media (75 μl) and incubated with 25 μlof a 4× serial dilution of each ADC and naked antibody prepared in cellculture media were added at concentrations of 0.013, 0.133, 1.33, 13.3,133, 266, and 633 nM for Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18 andPhor18-V_(L)-scFvFc and 0.013-266 nM for the naked antibody scFv-Fc andscFvFc-C_(H)3-Phor18.

Cells incubated for 4 hours were assayed for membrane integrity using aluminometric assay kit (Promega, Madison, Wis., Cytotox Glo G9292 lot#26229601). Cell viability was determined was determined after 24, and48 hours using a luminescent assay kit (Promega, Madison, Wis., CellTiter Glo, G 7572, lot 31386501). Controls for 100% cell viability(culture media) and 100% cell death (0.1% Triton X 100) incubated underthe same conditions.

Data were processed and analyzed to obtain IC50 values using Graph PadPrizm version 5.00 for Windows, GraphPad Software, San Diego Calif. USA,www-graphpad-com (Graph Pad Prizm, Inc). Statistical analysis forsignificance was determined by a two-tailed Student's T-test. Each testwas conducted using 2 plates with 2-3 wells each to achieve an N of 4-6data points per time point.

Recombinant ScFv-Fc-Phor18 conjugates were expressed in Pichia Pastoris.As shown in FIG. 13 and Table 13, the anti-CD20-Phor18 conjugatesdestroyed membrane integrity in CD20 positive Daudi cells after 4 h withIC₅₀ values of 6.2±2 nM for Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18, and23.5±2.5 nM for Phor18-V_(L)-scFvFc and 282.4±15 nM forscFv-Fc-C_(H)3-Phor18; unconjugated, naked scFvFc was not toxic. HumanBurkitts lymphoma cells (Daudi) were killed within 24 h with IC₅₀ valuesof 9.9±2 nM for Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18, and 18.8±4 nM forPhor18-V_(L)-scFvFc and 141.1±6 nM for scFv-Fc-C_(H)3-Phor18; nakedscFvFc had a IC₅₀ value of 287±28 nM. The lowest IC₅₀ values weremeasured after 48 hours with 1.6±0.3 nM forPhor18-V_(L)-scFv-Fc-C_(H)3-Phor18, and 3.4±0.05 nM forPhor18-V_(L)-scFvFc and 106±10 nM for scFv-Fc-C_(H)3-Phor18; nakedscFvFc had a IC₅₀ value of 339±26 nM. The CD20 negative human leukemiacells (U937) showed the same response to either conjugated andunconjugated ADC: after 4 hours no effects on membrane integrity, IC₅₀values after 24 hours were comparable to naked scFv-Fc with 298±13.4,246.1±14.8 for Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18, 875±81 forPhor18-V_(L)-scFvFc and 274±154 nM for scFv-Fc-C_(H)3-Phor18.

These data demonstrate that N-terminal conjugated ADCs are more potentthan C-terminal conjugated ADCs. ADCs with 4 Phor18 molecules were moreactive than conjugates with 2 Phor18 molecules.

TABLE 13 In vitro toxicities of Phor18-V_(L)-scFv-Fc andscFv-Fc-C_(H)3-Phor18, Phor18-V_(L)-scFv-Fc-C_(H)3-Phor18 and scFv-Fctargeting CD 20. Daudi cells (NHL) are positive for CD 20, U937 cells(leukemia) are negative for CD20. scFvFc Phor18-V_(L)-scFv-Fc-Phor18-V_(L)- scFv-Fc-C_(H)3- (naked) C_(H)3-Phor18 scFv-Fc Phor18Number of Phor18 0 4 (N- and C-terminus) 2 (N-terminus) 2 (C-terminus)IC₅₀ values IC₅₀ values [nM] IC₅₀ values [nM] [nM] IC₅₀ values [nM]Daudi 4 h (Membrane Not toxic 6.2 ± 2 23.5 ± 2.5 282.4 ± 15   integrity)24 h 287 ± 28 9.9 ± 2 18.8 ± 4   141.1 ± 6    48 h 339 ± 26   1.6 ± 0.3 3.4 ± 0.05 106 ± 10  U937 4 h (Membrane Not toxic Not toxic Not toxicNot toxic integrity) 24 h   298 ± 13.4  246.1 ± 14.8 875 ± 81 274 ± 15448 h 112.7 ± 4   117 ± 7  Not toxic 116 ± 4.5 

Potent scFv-Fc-Phor18 conjugates with 2 and 4 Phor18 molecules at the N-or C-terminus and N- and C-terminus were expressed in Pichia Pastoris.The ADCs were more potent than naked scFv-Fc. ScFv-Fc-Phor18 conjugatesdestroyed membrane integrity of the target cells when N and C-terminuswas conjugated or N-terminus was conjugated. C-terminus conjugation was50-100 fold less active compared to N-terminus and C- and N-terminusconjugated ADCs. CD20 targeted ADCs killed specifically target cells—thecell death was independent on internalization. Increasing numbers ofPhor18 on ADC resulted in increased potency.

Example 21

This example describes IgG2-Phor18 conjugates produced in mammaliansystem:

CHO cell expressed CD20 targeting ADCs represented whole antibodies andwere conjugated to 2, 4 and 6 Phor18 at different locations. Theproduced ADCs were characterized to confirm Phor18 presence on heavy andlight chains, their molecular weights.

Cytotoxicity was determined in vitro using the recombinantly producedCD20 targeting IgG2-Phor18 conjugates from a CHO cell expression systemand compared with “naked” antibody (Rituxan) in CD20 positive cells(Daudi, Burkitts lymphoma). CD20 negative leukemia cells (U937) servedas controls.

The V_(L) and V_(H) domains of the anti-CD20 ADC are humanized sequencesfrom anti-p185 Herceptin with the CDRs replaced by Rituxan anti-CD20CDRs. Whole human IgG2 was used for the full antibody backbone with theλ isoform of the constant light (C_(L)) domain.

The IgG2 isoform was chosen to minimize FcR interactions and limitbinding and killing of immune cells by Rituxan ADCs. Gene synthesis wasconducted by Genscript USA Inc., Piscataway, N.J., with codon usageoptimized for CHO cells. The amino acid sequences of the “unconjugated”anti-CD20 antibody Anti-CD20 antibody heavy (H) and light (L) chains areshown below. Rituxan CDRs in variable domains are shown in bold type.

Anti-CD20 Antibody Amino Acid Sequence of Light Chain and Heavy Chainwith Rituxan CDRs

Light chain: V_(L) (SEQ ID NO.: 105)        10         20         30         40         50         60MDIQMTQSPS SLSASVGDRV TITCRASSSV SYIHWYQQKP GKAPKLLIYA TSNLASGVPS        70         80         90        100RFSGSRSGTD FTLTISSLQP EDFATYYCQQ WTSNPPTFGQ GTKVEIKR C_(L) (λ)(SEQ ID NO.: 106)        10         20         30         40         50         60GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK        70         80         90        100QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS Heavy chain: V_(H)(SEQ ID NO.: 107)        10         20         30         40         50         60MEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ APGKGLEWVA AIYPGNGDTS        70         80         90        100        110        120YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS TYYGGDWYFD VWGQGTLVTV SSIGg2 C_(H)1 and hinge (SEQ ID NO.: 108)        10         20         30         40         50         60ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS        70         80         90        100        110GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCPIGg2 C_(H)2 and C_(H)3 (SEQ ID NO.: 109)        10         20         30         40         50         60APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP        70         80         90        100        110        120REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL       130        140        150        160        170        180PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT       190        200        210 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

Recombinant expression of whole IgG2 antibody-Phor18 (KFAKFAKKFAKFAKKFAK (SEQ ID NO.:4)) conjugates in a mammalian system (CHO cells). Heavyand light chain transcripts were synthesized out of the Genscript DNAtranscripts using PCR primers with Asc 1 and EcoR1 restriction sites fordirectional cloning into the pSECTag2 mammalian expression plasmid(Invitrogen cat# V900-20, lot 842626). The pSecTag2 plasmid was chosenbecause it has a mammalian CMV promoter and an Igk signal sequence forsecretion of the antibody. Because of the location of the multiplecloning site in the expression plasmid relative to the signal sequence,cleavage of the signal peptide in the expressed ADC protein leaves thefollowing 6 amino acids on the N-terminus of each peptide because of theplasmid design: DAAQPA (SEQ ID NO.:152).

Genscript ADC transcript DNA sequences are shown below for the fulllight chain and full heavy chain of the ADC with Phor18 (italics):

Sequence of the gene for expression of CD20 targeting antibody-Phor8conjugates in CHO cells

The full ADC light chain target sequence is: (SEQ ID NO.: 110)

GGTCAGATATTCAGATGACTCAGAGCCCCAGCTCCCTGTCCGCATCTGTGGGCGACCGAGTCACTATCACCTGCCGAGCCTCTAGTTCAGTGAGCTACATTCACTGGTATCAGCAGAAGCCTGGGAAAGCCCCAAAGCTGCTCATCTACGCCACAAGCAACCTGGCTTCCGGTGTGCCTTCTAGGTTCAGTGGGTCAAGAAGCGGTACAGACTTTACACTGACTATTAGCTCCCTCCAGCCAGAGGATTTCGCCACTTACTATTGCCAGCAGTGGACTTCCAATCCCCCTACCTTTGGCCAGGGAACAAAAGTGGAAATCAAGGGGCAGCCCAAAGCTAACCCTACCGTCACACTGTTCCCACCCTCTAGTGAGGAACTCCAGGCAAATAAGGCCACTCTGGTGTGTCTCATTTCCGACTTTTACCCCGGAGCTGTGACCGTCGCTTGGAAGGCAGATGGCTCTCCAGTGAAAGCAGGAGTCGAGACCACAAAACCCAGTAAGCAGTCAAACAATAAGTACGCCGCTTCAAGCTATCTGAGTCTCACCCCTGAACAGTGGAAAAGCCATAGGTCCTATTCTTGCCAGGTCACTCACGAAGGTAGCACTGTGGAAAAGACTGTCGCACCAACCGAATGTAGCGGCTCCAAGGGCTCCAAG

The full ADC heavy chain target sequence is: (SEQ ID NO.: 111)

GCTCCGAAGTGCAGCTCGTCGAAAGCGGGGGGGGACTCGTGCAGCCAGGGGGAAGCCTCAGACTCTCATGCGCCGCCTCAGGTTATACTTTCACAAGCTACAACATGCACTGGGTCAGACAGGCACCTGGGAAGGGTCTGGAGTGGGTGGCCGCTATCTACCCAGGCAACGGAGACACATCTTATAATCAGAAGTTCAAAGGCCGGTTTACTATTAGCGCAGATACATCCAAGAACACTGCCTACCTGCAGATGAATAGCCTCCGGGCTGAAGACACTGCAGTGTACTATTGCAGTCGCTCAACCTACTATGGCGGAGACTGGTATTTCGATGTGTGGGGGCAGGGTACTCTGGTCACCGTGAGCTCCGCCTCTACCAAGGGGCCCAGTGTGTTTCCACTGGCTCCCTGCAGCCGGTCCACCTCTGAGAGTACAGCAGCCCTGGGTTGTCTCGTGAAAGATTACTTCCCTGAACCAGTCACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTCCACACATTTCCTGCAGTGCTCCAGTCTAGTGGGCTGTACTCCCTCTCAAGCGTGGTCACAGTCCCATCCTCTAATTTCGGTACTCAGACCTATACATGCAACGTGGACCATAAGCCCTCCAATACTAAGGTCGATAAAACCGTGGAGCGCAAATGCTGTGTGGAATGCCCACCTTGTCCAGCACCACCAGTCGCTGGGCCTAGCGTGTTCCTGTTTCCTCCAAAGCCAAAAGACACTCTCATGATCTCTCGAACTCCCGAGGTCACCTGTGTGGTCGTGGACGTCAGTCACGAGGATCCTGAAGTCCAGTTTAACTGGTACGTGGATGGAGTCGAAGTGCATAATGCAAAGACCAAACCAAGGGAGGAACAGTTCAACTCAACCTTTAGAGTCGTGAGCGTGCTGACAGTCGTGCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGTCTGCCCGCTCCTATCGAGAAAACAATTAGCAAGACTAAAGGACAGCCTCGAGAACCACAGGTGTACACACTGCCCCCTAGCAGGGAGGAAATGACAAAGAACCAGGTCTCCCTGACTTGTCTCGTGAAAGGCTTCTATCCCAGTGACATTGCCGTGGAGTGGGAATCAAATGGACAGCCTGAGAACAATTACAAGACCACACCACCCATGCTGGACAGTGATGGCTCATTCTTTCTGTATTCCAAGCTCACCGTGGATAAATCTAGGTGGCAGCAGGGAAATGTCTTTTCATGTAGCGTGATGCACGAGGCTCTCCATAACCATTACACCCAGAAGTCCCTGTCACTCTCCCCCGGCAAAGGCTCCAAGG

TGATAAPCR primers used to subclone the light chains and heavy chains are shownbelow.ADC PCR Primers:Forward primers have the ASCI restriction site (GGCGCGCC) at the 5′ end.Reverse primers have the EcoR1 restriction site (GAATTC) at the 5′ end.

Light chain primers  (SEQ ID NOs.: 112-115) 480L for: GGGGGCGCGCC GATATTCAGATGACTCAGAGCC (Tm = 55.6) 485Lfor: GGGGGCGCGCC AAGTTCGCAAAGTTCGCCAA (Tm = 63) 480Lrev: GGG GAATTC TTATCAGCTACATTCGGTTGGT (Tm = 58.65) 487Lrev: GGG GAATTC TTATCATTTAAAGGCCTTGAATGCT (Tm = 61.37)  Mar18Heavy chain primers (SEQ ID NOs.: 116-118) 480Hfor: GGG GGCGCGCC GAAGTGCAGCTCGTCGAAAG (Tm = 61) 485Hfor: GGG GGCGCGCC AAGTTCGCCAAATTTGCTAAGA (Tm = 60.25) 480Hrev: GGG GAATTC TTATCATTTGCCGGGGGA (Tm = 62)

The amino acid sequences for each of the naked IgG2 antibody and thePhor18-IgG2 conjugates with 2 Phor18 molecules (Phor18-V_(L) IgG2,Phor18-V_(H) IgG2), 4 Phor18 molecules (Phor18-V_(L)-Phor18V_(H)-IgG2),6 Phor18 molecules (Phor18-V_(L)-C_(L)-Phor18-Phor18-V_(H)-IgG2) and 8Phor18 moleculesPhor18-V_(L)-C_(L)-Phor18-V_(H)-Phor18-C_(H)3-Phor18-IgG2 andPhor18-V_(L)-C_(L)-Phor18-V_(H)-Phor18-C_(H)3-IgG2) are shown below.

Amino acid sequences of naked antibody, lytic-peptide Phor18-antibodyheavy and light chain conjugates IgG2 (480) (naked) (SEQ ID NO.: 119)Light chain        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG KAPKLLIYAT SNLASGVPSR        70         80         90        100        110        120FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG TKVEIKGQPK ANPTVTLFPP       130        140        150        160        170        180SSEELQANKA TLVCLISDFY PGAVTVAWKA DGSPVKAGVE TTKPSKQSNN KYAASSYLSL       190        200        210 TPEQWKSHRS YSCQVTHEGS TVEKTVAPTE CSHeavy chain (SEQ ID NO.: 120)        10         20         30         40         50         60EVQLVESGGG LVQPGGSLRL SCAASGYTFT SYNMHWVRQA PGKGLEWVAA IYPGNGDTSY        70         80         90        100        110        120NQKFKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRST YYGGDWYFDV WGQGTLVTVS       130        140        150        160        170        180SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS       190        200        210        220        230        240SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKCCVECPPC PAPPVAGPSV       250        260        270        280        290        300FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD GVEVENAKTK PREEQFNSTF       310        320        330        340        350        360RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK       370        380        390        400        410        420NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG       430        440 NVFSCSVMHE ALHNHYTQKS LSLSPGKPhor18-V_(L) IgG2 (481) (2 Phor18, N-terminus) (SEQ ID NO.: 121)Light chain        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SDIQMTQSPS SLSASVGDRV TITCRASSSV SYIHWYQQKP        70         80         90        100        110        120GKAPKLLIYA TSNLASGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ WTSNPPTFGQ       130        140        150        160        170        180GTKVEIKGQP KANPTVTLFP PSSEELQANK ATLVCLISDF YPGAVTVAWK ADGSPVKAGV       190        200        210        220        230        240ETTKPSKQSN NKYAASSYLS LTPEQWKSHR SYSCQVTHEG STVEKTVAPT ECSHeavy chain (SEQ ID NO.: 122)        10         20         30         40         50         60EVQLVESGGG LVQPGGSLRL SCAASGYTFT SYNMHWVRQA PGKGLEWVAA IYPGNGDTSY        70         80         90        100        110        120NQKFKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRST YYGGDWYFDV WGQGTLVTVS       130        140        150        160        170        180SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS       190        200        210        220        230        240SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKCCVECPPC PAPPVAGPSV       250        260        270        280        290        300FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD GVEVENAKTK PREEQFNSTF       310        320        330        340        350        360RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK       370        380        390        400        410        420NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG       430        440 NVFSCSVMHE ALHNHYTQKS LSLSPGKPhor18-V_(H) IgG2(483)(2 Phor18, N-terminus)(SEQ ID NO.: 123)Light chain        10         20         30         40         50         60DIQMTQSPSS LSASVGDRVT ITCRASSSVS YIHWYQQKPG KAPKLLIYAT SNLASGVPSR        70         80         90        100        110        120FSGSRSGTDF TLTISSLQPE DFATYYCQQW TSNPPTFGQG TKVEIKGQPK ANPTVTLFPP       130        140        150        160        170        180SSEELQANKA TLVCLISDFY PGAVTVAWKA DGSPVKAGVE TTKPSKQSNN KYAASSYLSL       190        200        210 TPEQWKSHRS YSCQVTHEGS TVEKTVAPTE CSHeavy chain (SEQ ID NO.: 124)        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ        70         80         90        100        110        120APGKGLEWVA AIYPGNGDTS YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS       130        140        150        160        170        180TYYGGDWYFD VWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT       190        200        210        220        230        240VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV       250        260        270        280        290        300ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV       310        320        330        340        350        360DGVEVENAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT       370        380        390        400        410        420KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD       430        440        450        460SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKPhor18-17_(L)-Phor18V_(H)-IgG2(485)(4Phor18,2N-terminus)(SEQ ID NO.: 125)Light chain        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SDIQMTQSPS SLSASVGDRV TITCRASSSV SYIHWYQQKP        70         80         90        100        110        120GKAPKLLIYA TSNLASGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ WTSNPPTFGQ       130        140        150        160        170        180GTKVEIKGQP KANPTVTLFP PSSEELQANK ATLVCLISDF YPGAVTVAWK ADGSPVKAGV       190        200        210        220        230ETTKPSKQSN NKYAASSYLS LTPEQWKSHR SYSCQVTHEG STVEKTVAPT ECSHeavy chain (SEQ ID NO.: 126)        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ        70         80         90        100        110        120APGKGLEWVA AIYPGNGDTS YNQKFKGRFT ISADTSENTA YLQMNSLRAE DTAVYYCSRS       130        140        150        160        170        180TYYGGDWYFD VWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VEDYFPEPVT       190        200        210        220        230        240VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV       250        260        270        280        290        300ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV       310        320        330        340        350        360DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT       370        380        390        400        410        420KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD       430        440        450        460SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKPhor18-V_(L)-C_(L)-Phor18-Phor18-V_(H)-IgG2(487)(6 Phor18, 2 N and 1 C-terminus) (SEQ ID NO.: 127) Light chain        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SDIQMTQSPS SLSASVGDRV TITCRASSSV SYIHWYQQKP        70         80         90        100        110        120GKAPKLLIYA TSNLASGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ WTSNPPTFGQ       130        140        150        160        170        180GTKVEIKGQP KANPTVTLFP PSSEELQANK ATLVCLISDF YPGAVTVAWK ADGSPVKAGV       190        200        210        220        230        240ETTKPSKQSN NKYAASSYLS LTPEQWKSHR SYSCQVTHEG STVEKTVAPT ECSGSKAFKK       250 AFKAFKKAFK AFK Heavy chain (SEQ ID NO.: 128)        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ        70         80         90        100        110        120APGKGLEWVA AIYPGNGDTS YNQKFKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCSRS       130        140        150        160        170        180TYYGGDWYFD VWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VEDYFPEPVT       190        200        210        220        230        240VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV       250        260        270        280        290        300ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV       310        320        330        340        350        360DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT       370        380        390        400        410        420KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD       430        440        450        460SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKPhor18-V_(L)-C_(L)-Phor18-V_(H)-Phor18-C_(H)3-Phor18-IgG2(489)(8 Phor18, 2 N and 2 C-terminus) (SEQ ID NO.: 129) Light chain        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SDIQMTQSPS SLSASVGDRV TITCRASSSV SYIHWYQQKP        70         80         90        100        110        120GKAPKLLIYA TSNLASGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ WTSNPPTFGQ       130        140        150        160        170        180GTKVEIKGQP KANPTVTLFP PSSEELQANK ATLVCLISDF YPGAVTVAWK ADGSPVKAGV       190        200        210        220        230        240ETTKPSKQSN NKYAASSYLS LTPEQWKSHR SYSCQVTHEG STVEKTVAPT ECSGSKAFKK       250 AFKAFKKAFK AFK Heavy chain (SEQ ID NO.: 130)        10         20         30         40         50         60MKFAKFAKKF AKFAKKFAKG SEVQLVESGG GLVQPGGSLR LSCAASGYTF TSYNMHWVRQ        70         80         90        100        110        120APGKGLEWVA AIYPGNGDTS YNQKFKGRFT ISADTSENTA YLQMNSLRAE DTAVYYCSRS       130        140        150        160        170        180TYYGGDWYFD VWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VEDYFPEPVT       190        200        210        220        230        240VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV       250        260        270        280        290        300ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV       310        320        330        340        350        360DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT       370        380        390        400        410        420KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD       430        440        450        460        470        480SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGS KAFKKAFKAFKKAFKAFK

Invitrogen free-style suspension grown CHO cells (Free-style MAX CHOexpression system cat# K9000-20) were transfected using Invitrogenprotocols. Briefly, FS CHO cells were expanded for 6 to 7 days afterthawing to rapid growth phase, doubling every 24 h. The day beforetransfection, clumps were removed, cells were pelleted and resuspendedin P/S-free medium at 5×10⁵/ml. On the day of transfection, 30 mlcultures of cells were adjusted to 9×10⁵/ml if necessary and viabilityshould be close to 99%.

Each 125 ml spinner flask (VWR cat# PBV 125) of 30 ml cells wastransfected with 35 μg of total plasmid DNA mixed with 35 μl of FSMaxtransfection reagent (Invitrogen cat#16447-100). DNA should be at 1mg/ml or higher in concentration. Cells were swirled rapidly whileadding DNA mixture slowly. Ratios of H:L chains tested were 3:2, 1:1,and 2:3.

Protein was harvested on day 3 and day 6 after transfection.Approximately 0.25 ml of protein A resin (Genscript L00210, capacity >20mg IgG per ml resin) was used to isolate secreted ADCs from the FS CHOmedium using the Genscript protocol provided The expressed anti-CD20IgG2 antibody (humanized variable light and heavy domains regions toCD20 receptor) and the various antibody-Phor18 conjugates withstoichiometric ratios of Phor18: AB of 2:1, 4:1 and 6:1 werecharacterized using SDS PAGE, and Western blot analyses (Table 14).

TABLE 14 ADC descriptions for CD20 targeting ADC Number of Name of ADCID Phor18 location Phor18/Antibody IgG2 480 None (‘Naked’) 0Phor18-V_(L)-IgG2 481 N-termini light chains (V_(L)) 2 Phor18-V_(H)-IgG2483 N-termini heavy chains (V_(H)) 2 Phor18-V_(L)-Phor18-V_(H)-IgG2 485N-termini heavy and light chains (V_(H), V_(L)) 4Phor18-V_(L)-Phor18-V_(H)-C_(L)- 487 N-termini and C-termini lightchains 6 Phor18-IgG2 (V_(L), V_(H), C_(L))Phor18-V_(L)-C_(L)-Phor18-V_(H)- 489 N-termini and C-termini lightchains 8 Phor18-C_(H)3-Phor18-IgG2 (V_(L), C_(L), V_(H), C_(H)3)

ADCs were probed on Western blots probed with anti-Phor18. All ADCs havea single Phor18 on the heavy and light chains. Yield, purity andcytotoxicity of recombinantly produced antibodies (as a full IgG2antibody) with Heavy (H) or Light (L) chain C-terminal- orN-terminal-Phor18 conjugation was analyzed. Two, 4 and 6 molecules oflytic domains (Phor18) conjugated to whole antibody molecule wereexpressed.

The concentration of recombinantly produced antibody and antibody-Phor18conjugates was determined using spectrophotometric measurements (OD₂₈₀)and were as follows: IgG2 (Mw 150,000; 1.096 mg/ml), Phor18-V_(L)-IgG2(Mw 154,340 g/mol, 0.561 mg/ml), Phor18-V_(H)-IgG2 (Mw 154,340 g/mol,0.1 mg/ml) and Phor18-V_(L)-Phor18V_(H)-IgG2 (Mw 158,400 g/mol, 0.561mg/ml) and Phor18-V_(L)-Phor18V_(H)-C_(L)-Phor18-IgG2 (Mw 162,600, 0.07mg/ml).

Example 22

This example describes in vitro activity of recombinantly producedIgG2-Phor18 conjugates.

The cytotoxicity of recombinantly produced CD20 targeting IgG2-Phor18conjugates in a mammalian expression system was compared with “naked”antibody (IgG2) in CD20 positive cells (Daudi, Burkitts lymphoma). CD20negative leukemia cells (U937) served as controls. CHO cell expressedADCs represented intact antibodies and were conjugated to 2, 4 and 6Phor18 molecules at the N-terminus of the V_(L) chain at the N-terminusof the V_(H) chain, at the N-terminus of the V_(H) and V_(L) chain andat the N-terminus at the V_(L), V_(H) and C-terminus of the C_(L) chain.The sequence descriptions are summarized in Table 14.

Human Non-Hodgkin's lymphoma cells Daudi (CD20 positive, passage numberp6) and human leukemia cell line U937 (CD20 negative, p 10) were seededat a density of 2,000 cells per well in opaque plates in heatinactivated full medium using cell dissociation buffer. After 24 hourscells were replenished with fresh media (75 μl) and incubated with 25 μlof a 4× serial dilution of each ADC and naked antibody prepared in cellculture media were added at concentrations between 0.001-200 nM forIgG2, Phor18-V_(L)-IgG2, Phor18-V_(H)-IgG2 andPhor18-V_(L)-Phor18V_(H)-IgG2, and 0.001-100 nM forPhor18-V_(L)-Phor18V_(H)-CL-Phor18-IgG2.

Cells incubated for 4 hours were assayed for membrane integrity using aluminometric assay kit (Promega, Madison, Wis., Cytotox Glo G9292 lot#317872). Cell viability was determined was determined after 24, and 48hours using a luminescent assay kit (Promega, Madison, Wis., Cell TiterGlo, G 7572, lot 336262).

Controls for 100% cell viability (culture media) and 100% cell death(0.1% Triton X 100) incubated under the same conditions.

Data were processed and analyzed to obtain IC50 values using Graph PadPrizm version 5.00 for Windows, GraphPad Software, San Diego Calif. USA,www-graphpad-com (Graph Pad Prizm, Inc). Statistical analysis forsignificance was determined by a two-tailed Student's T-test. Each testwas conducted using double plates of 2-3 wells each to achieve an N of4-6 data points per time point.

TABLE 15 In vitro toxicities of anti-CD20 IgG2, Phor18-V_(L)-IgG2,Phor18-V_(H)-IgG2, Phor18-V_(L)- Phor18V_(H)-IgG2 andPhor18-V_(L)-Phor18V_(H)-C_(L)-Phor18-IgG2, targeting CD 20. Daudi cells(NHL) were positive for CD 20, U937 cells (leukemia) are negative forCD20. Phor18-V_(L)- Phor18-V_(L)- Phor18-V_(L)-Phor18V_(H)-C_(L)- IgG2Phor18-V_(H)- Phor18V_(H)-IgG2 Phor18-IgG2 IgG2 IC₅₀ [nM] IgG2 IC₅₀ [nM]IC₅₀ [nM] naked V_(L)N- IC₅₀ [nM] V_(L) and V_(H)N- V_(L) andV_(H)N-terminus, C_(L) 0 terminus V_(H)N-terminus terminus C-terminusIC₅₀ 2 Phor18 2 Phor18 4 Phor18 6 Phor18 [nM] IC₅₀ [nM] IC₅₀ [nM] IC₅₀[nM] IC₅₀ [nM] Daudi 4 h Not ND 1108 ± 198 109.6 ± 20.1 71.6 ± 29 (Membrane toxic integrity) 24 h Not 824 ± 63 75.24 ± 37.5  40.1 ± 15.118.8 ± 5.9 toxic 48 h Not  >436 4.8 ± 2  20.4 ± 8.2  1.9 ± 0.2 toxicU937 4 h (Membrane Not Not toxic Not toxic Not toxic Not toxicintegrity) toxic 24 h Not Not toxic Not toxic Not toxic Not toxic toxic48 h Not >1000 Not toxic 249.6 ± 29   258 ± 41 toxic ND = not determined

Destruction of membrane integrity was measured after 4 hours atnanomolar concentrations of 1,108±198 for Phor18-V_(H)-IgG2 (2 Phor18),109.6±20.1 for Phor18-V_(L)-Phor18V_(H)-IgG2 (4 Phor18) and 71.6±29 forPhor18-V_(L)-Phor18V_(H)-C_(L)-Phor18-IgG2 (6 Phor18). Naked antibodywas not toxic after 24 or 48 hours to the CD20 positive Daudi cells.

Killing of the target cells after 24 h expressed as IC₅₀ values [nM]were 824±63, 75.24±37.5, (2 Phor18), 40.1±15.1 (4 Phor18) and 18.8±5.9(6 Phor18) nM. Maximal effects measured after 48 h (IC50 [nM]>436(Phor18-V_(L)-IgG2), 4.8±2 Phor18-V_(H)-IgG2 (2 Clips) and 20.4±8.2 nMPhor18-V_(L)-Phor18V_(H)-IgG2 (4 Clips), and 1.9±0.2 nMPhor18-V_(L)-Phor18V_(H)-C_(L)-Phor18-IgG2 (6 Phor18) respectively). TheCD20 negative cell line (U937) was not killed by either naked antibodiesor both 2 4, and 6 Phor18 conjugated ADCs after 4 and 24 hours. Toxicitylevels after 48 hours were similar with IC₅₀ values of 249 and 258 nM.These data demonstrate that position of Phor18 determines the potency ofa CD20 ADC with increased potency for Phor18-V_(H)-IgG2. Increase ofPhor18 molecules per antibody increased the potency.

Whole antibody Phor18 conjugates with Phor18:antibody stoichiometries of2, 4 and 6 were recombinantly expressed in CHO cells. These antibodydrug conjugates had confirmed Phor18 molecules at heavy and lightchains. They were active in CD20 positive cells in vitro with activitiesof membrane disintegration after 4 hours. After 48 hours single digitnanomolar activities were measured that were highest in ADC with 6 and 4Phor18.

Non-internalizing CD20 antibody conjugates destroyed membrane integrityof target cells (Daudi) within 4 h. Cell death was observed after 24 hwith IC₅₀ values in the low nanomolar range. CD20 negative cells (U937)were not killed. Full IgG2 ADCs having 4 and 6 clips had increasedpotency to target positive cells compared to 2 Phor18 conjugates. Thepositioning of Phor18 on the N-terminus generated ADCs with higherpotencies. Increase of Phor18 numbers per antibody showed increasedpotencies.

Higher numbers of Phor18 (six vs four vs two) on the antibody conjugatedto the N-terminal domain are more potent compared to naked antibodies.

Example 23

This example includes target sequence information for representativetarget proteins to which antibody and polypeptide conjugates of theinvention can be produced and naked antibodies and their Phor18conjugates

ERBB2 (HER2/NEU) Isoform 1 [UniParc]: (SEQ ID NO.: 131)        10         20         30         40         50        60MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL        70         80         90        100        110        120ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG       130        140        150        160        170        180DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA       190        200        210        220        230        240LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC       250        260        270        280        290        300AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP       310        320        330        340        350        360YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN       370        380        390        400        410        420IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP       430        440        450        460        470        480DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV       490        500        510        520        530        540PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC       550        560        570        580        590        600VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC       610        620        630        640        650        660PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG       670        680        690        700        710        720ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETEL       730        740        750        760        770        780RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDE AYVMAGVGSP       790        800        810        820        830        840YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR       850        860        870        880        890        900LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT       910        920        930        940        950        960HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM       970        980        990       1000       1010       1020IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA      1030       1040       1050       1060       1070       1080EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG      1090       1100       1110       1120       1130       1140AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV      1150       1160       1170       1180       1190       1200NQPDVRPQPP SPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ      1210       1220       1230       1240       1250GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPV

CD19:

B-lymphocyte surface antigen B4, component of the B-cell co-receptor,NCBI Reference Sequence: NP_001171569.1:

ORIGIN (SEQ ID NO.: 132)   1mppprllffl lfltpmevrp eeplvvkvee gdnavlqclk gtsdgptqql twsresplkp  61flklslglpg lgihmrplai wlfifnvsqq mggfylcqpg ppsekawqpg wtvnvegsge 121lfrwnvsdlg glgcglknrs segpsspsgk lmspklyvwa kdrpeiwege ppclpprdsl 181nqsIsqdltm apgstlwlsc gvppdsysrg plswthvhpk gpksllslel kddrpardmw 241vmetglllpr ataqdagkyy chrgnitmsf hleitarpvl whwllrtggw kvsavtlayl 301ifclcslvgi lhlqralvlr rkrkrmtdpt rrffkvtppp gsgpqnqygn vlslptptsg 361lgraqrwaag lggtapsygn pssdvqadga lgsrsppgvg peeeegegye epdseedsef 421yendsnlgqd qlsqdgsgye npedeplgpe dedsfsnaes yenedeeltq pvartmdfls 481phgsawdpsr eatslagsqs yedmrgilya apqlrsirgq pgpnheedad syenmdnpdg 541pdpawggggr mgtwstr

CD20:

a type III transmembrane protein found on B cells that forms a calciumchannel in the cell membrane allowing for the influx of calcium requiredfor cell activation; expressed in B-cell lymphomas, hairy cell leukemia,and B-cell chronic lymphocytic leukemia. Important for therapy of thosediseases, as an antibody against CD20 exists: Rituximab. NCBI ReferenceSequence

NP_068769.2, MS4A1-P11836: (SEQ ID NO.: 133)   1mttprnsvng tfpaepmkgp iamqsgpkpl frrmsslvgp tqsffmresk tlgavqimng  61lfhialggll mipagiyapi cvtvwyplwg gimyiisgsl laateknsrk clvkgkmimn 121slslfaaisg milsimdiln ikishflkme slnfirahtp yiniyncepa npseknspst 181qycysiqslf lgilsvmlif affqelviag ivenewkrtc srpksnivIl saeekkeqti 241eikeevvglt etssqpknee dieiipiqee eeeetetnfp eppqdqessp iendssp

CD22:

a sugar binding transmembrane protein that specifically binds sialicacid with an immunoglobulin (Ig) domain located at its N-terminus. It isa member of the immunoglobulin superfamily and the SIGLEC family. CD22functions as an inhibitory receptor for B cell receptor (BCR) signaling.NCBI Reference Sequence NP_001172028.1:

ORIGIN (SEQ ID NO.: 134)   1mhllgpwlll lvleylafsd sskwvfehpe tlyawegacv wipctyrald gdlesfilfh  61npeynkntsk fdgtrlyest kdgkvpseqk rvqflgdknk nctlsihpvh lndsgqlglr 121mesktekwme rihlnvserp fpphiqlppe iqesqevtlt cllnfscygy piqlqwlleg 181vpmrqaavts tsltiksvft rselkfspqw shhgkivtcq lqdadgkfls ndtvqlnvkh 241tpkleikvtp sdaivregds vtmtcevsss npeyttvswl kdgtslkkqn tftlnlrevt 301kdqsgkyccq vsndvgpgrs eevflqvqyp pkkvttviqn pmpiregdtv tlscnynssn 361psvtryewkp hgaweepslg vlkiqnvgwd nttiacaacn swcswaspva lnvqyaprdv 421rvrkikplse ihsgnsyslq cdfssshpke vqffwekngr llgkesqlnf dsispedags 481yscwvnnsig qtaskawtle vlyaprrlry smspgdqvme gksatltces danppvshyt 541wfdwnnqslp yhsqklrlep vkvqhsgayw cqgtnsvgkg rsplstltvy yspetigrry 601avglgsclai lilaicglkl qrrwkrtqsq qglqenssgq sffvrnkkvr raplsegphs 661lgcynpmmed gisyttlrfp emniprtgda essemqrppp dcddtvtysa lhkrqvgdye 721nvipdfpede gihyseliqf gvgerpqaqe nvdyvilkh

CD23:

a type II transmembrane protein found on mature B cells, monocytes,activated macrophages, eosinophils, platelets, and dendritic cells thatenhances capture and processing of antigen complexed with IgE. NCBIReference Sequence NP_001193948.2:

ORIGIN (SEQ ID NO.: 135)   1mnppsgelee lprrrccrrg tqivllglvt aalwaglltl lllwhwdttq slkqleeraa  61rnvsqvsknl eshhgdqmaq ksqstqisqe leelraeqqr lksqdlelsw nlnglqadls 121sfksqelner neasdllerl reevtklrme lqvssgfvcn tcpekwinfq rkcyyfgkgt 181kqwvharyac ddmegqlvsi hspeeqdflt khashtgswi glrnldlkge fiwvdgshvd 241ysnwapgept srsqgedcvm mrgsgrwnda fcdrklgawv cdrlatctpp asegsaesmg 301pdsrpdpdgr lptpsaplhs //

CD27:

TNF-receptor. Present on the surface of resting memory B cells. NCBIReference Sequence NP_001233.1:

ORIGIN (SEQ ID NO.: 136)   1marphpwwlc vlgtivglsa tpapkscper hywaqgklcc qmcepgtflv kdcdqhrkaa  61qcdpcipgvs fspdhhtrph cescrhcnsg llvrnctita naecacrngw qcrdkectec 121dplpnpslta rssqalsphp qpthlpyvse mleartaghm qtladfrqlp artlsthwpp 181qrslcssdfi rilvifsgmf lvftlagalf lhqrrkyrsn kgespvepae peryscpree 241egstipiqed yrkpepacsp

CD28:

present on all T-cells, and when matched with the appropriate ligand,labeled B7 which can be either CD80 or CD86, it has costimulatory effecton the T-cell. It is also expressed on Eosinophil granulocytes,especially after tissue infiltration. There its ligation leads torelease of potent neurotoxins, IL-2 and IL-13 as well as IFN-γ. Checksum1D9B6552A5878DOF: (SEQ ID NO.: 137)

        10         20         30         40         50         60MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD        70         80         90        100        110        120SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP       130        140        150        160        170        180PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR       190        200        210        220SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS

CD30:

a type I transmembrane protein present on activated T and B cells thatmay play a role in cell activation and/or differentiation; expressed inHodgkin disease, some T-cell lymphomas, and anaplastic large celllymphomas. (SEQ ID NO.:138)

        10         20         30         40         50         60MRVLLAALGL LFLGALRAFP QDRPFEDTCH GNPSHYYDKA VRRCCYRCPM GLFPTQQCPQ        70         80         90        100        110        120RPTDCRKQCE PDYYLDEADR CTACVTCSRD DLVEKTPCAW NSSRVCECRP GMFCSTSAVN       130        140        150        160        170        180SCARCFFHSV CPAGMIVKFP GTAQKNTVCE PASPGVSPAC ASPENCKEPS SGTIPQAKPT       190        200        210        220        230        240PVSPATSSAS TMPVRGGTRL AQEAASKLTR APDSPSSVGR PSSDPGLSPT QPCPEGSGDC       250        260        270        280        290        300RKQCEPDYYL DEAGRCTACV SCSRDDLVEK TPCAWNSSRT CECRPGMICA TSATNSCARC       310        320        330        340        350        360VPYPICAAET VTKPQDMAEK DTTFEAPPLG TQPDCNPTPE NGEAPASTSP TQSLLVDSQA       370        380        390        400        410        420SKTLPIPTSA PVALSSTGKP VLDAGPVLFW VILVLVVVVG SSAFLLCHRR ACRKRIRQKL       430        440        450        460        470        480HLCYPVQTSQ PKLELVDSRP RRSSTQLRSG ASVTEPVAEE RGLMSQPLME TCHSVGAAYL       490        500        510        520        530        540ESLPLQDASP AGGPSSPRDL PEPRVSTEHT NNKIEKIYIM KADTVIVGTV KAELPEGRGL       550        560        570        580        590AGPAEPELEE ELEADHTPHY PEQETEPPLG SCSDVMLSVE EEGKEDPLPT AASGK

P28908-2, Tumor necrosis factor receptor superfamily member 8, Homosapiens (SEQ ID NO.: 139):

        10         20         30         40         50         60MSQPLMETCH SVGAAYLESL PLQDASPAGG PSSPRDLPEP RVSTEHTNNK IEKIYIMKAD        70         80         90        100        110        120TVIVGTVKAE LPEGRGLAGP AEPELEEELE ADHTPHYPEQ ETEPPLGSCS DVMLSVEEEG       130 KEDPLPTAAS GK

CD31:

PECAM-1, a cell adhesion molecule on platelets and endothelial cells(SEQ ID NO.: 140).

        10         20         30         40         50         60MQPRWAQGAT MWLGVLLTLL LCSSLEGQEN SFTINSVDMK SLPDWTVQNG KNLTLQCFAD        70         80         90        100        110        120VSTTSHVKPQ HQMLFYKDDV LFYNISSMKS TESYFIPEVR IYDSGTYKCT VIVNNKEKTT       130        140        150        160        170        180AEYQLLVEGV PSPRVTLDKK EAIQGGIVRV NCSVPEEKAP IHFTIEKLEL NEKMVKLKRE       190        200        210        220        230        240KNSRDQNFVI LEFPVEEQDR VLSFRCQARI ISGIHMQTSE STKSELVTVT ESFSTPKFHI       250        260        270        280        290        300SPTGMIMEGA QLHIKCTIQV THLAQEFPEI IIQKDKAIVA HNRHGNKAVY SVMAMVEHSG       310        320        330        340        350        360NYTCKVESSR ISKVSSIVVN ITELFSKPEL ESSFTHLDQG ERLNLSCSIP GAPPANFTIQ       370        380        390        400        410        420KEDTIVSQTQ DFTKIASKSD SGTYICTAGI DKVVKKSNTV QIVVCEMLSQ PRISYDAQFE       430        440        450        460        470        480VIKGQTIEVR CESISGTLPI SYQLLKTSKV LENSTKNSND PAVFKDNPTE DVEYQCVADN       490        500        510        520        530        540CHSHAKMLSE VLRVKVIAPV DEVQISILSS KVVESGEDIV LQCAVNEGSG PITYKFYREK       550        560        570        580        590        600EGKPFYQMTS NATQAFWTKQ KASKEQEGEY YCTAFNRANH ASSVPRSKIL TVRVILAPWK       610        620        630        640        650        660KGLIAVVIIG VIIALLIIAA KCYFLRKAKA KQMFVEMSRF AVFLLNSNNE KMSDFNMEAN       670        680        690        700        710        720SHYGHNDDVR NHAMKPINDN KEPLNSDVQY TEVQVSSAES HKDLGKKDTE TVYSEVRKAV       730 PDAVESRYSR TEGSLDGT

CD33:

a marker of unknown function found on immature myeloid cells, includingacute myeloid leukemia blasts and mature monocytes. P20138: (SEQ IDNO.:141)

        10         20         30         40         50         60MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW        70         80         90        100        110        120FREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM       130        140        150        160        170        180ERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWL       190        200        210        220        230        240SAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT       250        260        270        280        290        300GIFPGDGSGK QETRAGVVHG AIGGAGVTAL LALCLCLIFF IVKTHRRKAA RTAVGRNDTH       310        320        330        340        350        360PTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSEVRTQ

CD34:

stem cell marker, adhesion, found on hematopoietic precursors (found inhigh concentrations in umbilical cord blood), capillary endothelium, andembryonic fibroblasts. Isoform CD34-F: (SEQ ID NO.:142)

        10         20         30         40         50         60MLVRRGARAG PRMPRGWTAL CLLSLLPSGF MSLDNNGTAT PELPTQGTFS NVSTNVSYQE        70         80         90        100        110        120TTTPSTLGST SLHPVSQHGN EATTNITETT VKFTSTSVIT SVYGNTNSSV QSQTSVISTV       130        140        150        160        170        180FTTPANVSTP ETTLKPSLSP GNVSDLSTTS TSLATSPTKP YTSSSPILSD IKAEIKCSGI       190        200        210        220        230        240REVKLTQGIC LEQNKTSSCA EFKKDRGEGL ARVLCGEEQA DADAGAQVCS LLLAQSEVRP       250        260        270        280        290        300QCLLLVLANR TEISSKLQLM KKHQSDLKKL GILDFTEQDV ASHQSYSQKT LIALVTSGAL       310        320        330        340        350        360LAVLGITGYF LMNRRSWSPT GERLGEDPYY TENGGGQGYS SGPGTSPEAQ GKASVNRGAQ       370        380 ENGTGQATSR NGHSARQHVV ADTEL

CD40:

a costimulatory protein found on antigen presenting cells. CD40 combineswith CD154 (CD40L) on T cells to induce antibody isotype switching in Bcells. Isoform I: (SEQ ID NO.:143)

        10         20         30         40         50         60MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL        70         80         90        100        110        120PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV       130        140        150        160        170        180LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN       190        200        210        220        230        240KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD       250        260        270DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ

CD52:

P31358m(SEQ ID NO.:144)

        10         20         30         40         50         60MKRFLFLLLT ISLLVMVQIQ TGLSGQNDTS QTSSPSASSN ISGGIFLFFV ANAIIHLFCF S

Q9UJ81 (SEQ ID NO.: 145)

        10 MKRFLFLLLT ISLLVMVQ

CD 56:

Neural cell adhesion molecule 1. Short name=N-CAM-1, Alternativename(s). CD_antigen=CD56. Isoform 1: (SEQ ID NO.:146)

        10         20         30         40         50         60MLQTKDLIWT LFFLGTAVSL QVDIVPSQGE ISVGESKFFL CQVAGDAKDK DISWFSPNGE        70         80         90        100        110        120KLTPNQQRIS VVWNDDSSST LTIYNANIDD AGIYKCVVTG EDGSESEATV NVKIFQKLMF       130        140        150        160        170        180KNAPTPQEFR EGEDAVIVCD VVSSLPPTII WKHKGRDVIL KKDVRFIVLS NNYLQIRGIK       190        200        210        220        230        240KTDEGTYRCE GRILARGEIN FKDIQVIVNV PPTIQARQNI VNATANLGQS VTLVCDAEGF       250        260        270        280        290        300PEPTMSWTKD GEQIEQEEDD EKYIFSDDSS QLTIKKVDKN DEAEYICIAE NKAGEQDATI       310        320        330        340        350        360HLKVFAKPKI TYVENQTAME LEEQVTLTCE ASGDPIPSIT WRTSTRNISS EEKASWTRPE       370        380        390        400        410        420KQETLDGHMV VRSHARVSSL TLKSIQYTDA GEYICTASNT IGQDSQSMYL EVQYAPKLQG       430        440        450        460        470        480PVAVYTWEGN QVNITCEVFA YPSATISWFR DGQLLPSSNY SNIKIYNTPS ASYLEVTPDS       490        500        510        520        530        540ENDFGNYNCT AVNRIGQESL EFILVQADTP SSPSIDQVEP YSSTAQVQFD EPEATGGVPI       550        560        570        580        590        600LKYKAEWRAV GEEVWHSKWY DAKEASMEGI VTIVGLKPET TYAVRLAALN GKGLGEISAA       610        620        630        640        650        660SEFKTQPVQG EPSAPKLEGQ MGEDGNSIKV NLIKQDDGGS PIRHYLVRYR ALSSEWKPEI       670        680        690        700        710        720RLPSGSDHVM LKSLDWNAEY EVYVVAENQQ GKSKAAHFVF RTSAQPTAIP ANGSPTSGLS       730        740        750        760        770        780TGAIVGILIV IFVLLLVVVD ITCYFLNKCG LFMCIAVNLC GKAGPGAKGK DMEEGKAAFS       790        800        810        820        830        840KDESKEPIVE VRTEEERTPN HDGGKHTEPN ETTPLTEPEK GPVEAKPECQ ETETKPAPAE       850 VKTVPNDATQ TKENESKA

CD70:

Tumor necrosis factor ligand superfamily member 7. P32970 (CD70_HUMAN):(SEQ ID NO.:147)

        10         20         30         40         50         60MPEEGSGCSV RRRPYGCVLR AALVPLVAGL VICLVVCIQR FAQAQQQLPL ESLGWDVAEL        70         80         90        100        110        120QLNHTGPQQD PRLYWQGGPA LGRSELHGPE LDKGQLRIHR DGIYMVHIQV TLAICSSTTA       130        140        150        160        170        180SRHHPTTLAV GICSPASRSI SLLRLSFHQG CTIASQRLTP LARGDTLCTN LTGTLLPSRN       190 TDETFFGVQW VRP

CD123:

IL3RA. Isoform 1: (SEQ ID NO.:148)

        10         20         30         40         50         60MVLLWLTLLL IALPCLLQTK EDPNPPITNL RMKAKAQQLT WDLNRNVTDI ECVYDADYSM        70         80         90        100        110        120PAVNNSYCQF GAISLCEVTN YTVRVANPPF STWILFPENS GKPWAGAENL TCWIHDVDFL       130        140        150        160        170        180SCSWAVGPGA PADVQYDLYL NVANRRQQYE CLHYKTDAQG TRIGCRFDDI SRLSSGSQSS       190        200        210        220        230        240HILVRGRSAA FGIPCTDKFV VFSQIEILTP PNMTAKCNKT HSFMHWKMRS HFNRKFRYEL       250        260        270        280        290        300QIQKRMQPVI TEQVRDRTSF QLLNPGTYTV QIRARERVYE FLSAWSTPQR FECDQEEGAN       310        320        330        340        350        360TRAWRTSLLI ALGTLLALVC VFVICRRYLV MQRLFPRIPH MKDPIGDSFQ NDKLVVWEAG       370 KAGLEECLVT EVQVVQKT

CD154:

The ligand for CD40. This is a costimulatory molecule that plays manyroles, best known for activating B cells but also known to induce theactivation of an APC in association with T cell receptor stimulation byMHC molecules on the APC. Q3L8U2: (SEQ ID NO.:149)

        10         20         30         40         50         60MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLH        70         80         90        100        110        120EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKVLQWA       130        140        150        160        170        180EKGYYTMSNN LVTLENGKQL TVKRQGLYYI YAQVTFCSNR EASSQAPFIA SLCLKSPGRF       190        200        210        220        230        240ERILLRAANT HSSAKPCGQQ SIHLGGVFEL QPGASVFVNV TDPSQVSHGT GFTSFGLLKL

CD138:

syndecan, a plasma cell-surface glycoprotein, known as syndecan-1.Syndecan functions as the alpha receptor for collagen, fibronectin andthrombospondin. P18827: (SEQ ID NO.: 150)

        10         20         30         40         50         60MRRAALWLWL CALALSLQPA LPQIVATNLP PEDQDGSGDD SDNFSGSGAG ALQDITLSQQ        70         80         90        100        110        120TPSTWKDTQL LTAIPTSPEP TGLEATAAST STLPAGEGPI EGEAVVLPEV EPGLTAREQE       130        140        150        160        170        180ATPRPRETTQ LPTTHLASTT TATTAQEPAT SHPHRDMQPG HHETSTPAGP SQADLHTPHT       199        200        210        220        230        240EDGGPSATER AAEDGASSQL PAAEGSGEQD FTFETSGENT AVVAVEPDRR NQSPVDQGAT       250        260        270        280        290        300GASQGLLDRK EVLGGVIAGG LVGLIFAVCL VGFMLYRMKK KDEGSYSLEE PKQANGGAYQ       310 KPTKQEEFYA KPTKQEEFYA

Oncofetoprotein-5T4 Trophoblast Glycoprotein.

NCBI Reference Sequence NP_001159864.1:

ORIGIN (SEQ ID NO.: 151) 1mpggcsrgpa agdgrlrlar lalvllgwvs sssptssass fsssapflas aysaqpplpd 61qcpalcecse aartvkcvnr nltevptdlp ayvrnlfltg nqlavlpaga farrpplael 121aalnlsgsrl devragafeh lpslrqldls hnpladlspf afsgsnasvs apsplvelil 181nhivppeder qnrsfegmvv aallagralq glrrlelasn hflylprdvl aqlpslrhld 241lsnnslvslt yvsfrnlthl eslhlednal kvlhngtlae lqglphirvf ldnnpwvcdc 301hmadmvtwlk etevvqgkdr ltcaypekmr nrvllelnsa dldcdpilpp slqtsyvflg 361ivlaligaif llvlylnrkg ikkwmhnird acrdhmegyh yryeinadpr ltnlssnsdv

What is claimed:
 1. A method of reducing or inhibiting proliferation ofa cell, the method comprising contacting a cell with: (i) an antibodyconjugate comprising an antibody that binds to CD20 or CD19, wherein theantibody is linked to two or more lytic domains, wherein said lyticdomains comprise or consist of a peptide selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6,respectively), or a peptide selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6,respectively) having one or more of the F, A, or K residues of the lyticdomain substituted with a corresponding D-amino acid; wherein one ofsaid lytic domains is linked to the amino(NH2)-terminus of a Light (L)chain of the antibody, and the other of said lytic domains is linked tothe amino(NH2)-terminus of a Heavy (H) chain of the antibody; and (ii)an alkylating agent, anti-metabolite, plant extract, plant alkaloid,nitrosourea, hormone, nucleoside or nucleotide analog; thereby reducingor inhibiting proliferation of the cell.
 2. A method of reducing orinhibiting proliferation of a cell, the method comprising contacting acell with: (i) a polypeptide conjugate comprising a Heavy (H) chain anda Light (L) chain of an antibody that binds to CD20 or CD19, wherein theHeavy (H) and Light (L) chain variable regions each comprise 3 CDRs,wherein the amino(NH2)-terminus of the Light (L) chain is linked to alytic domain, wherein the amino(NH2)-terminus of the Heavy (H) chain islinked to a lytic domain, wherein said lytic domains comprise or consistof a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF andKFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6, respectively), or a peptideselected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ IDNOs.:1-6, respectively) having one or more of the F, A, or K residues ofthe lytic domain substituted with a corresponding D-amino acid; and (ii)an alkylating agent, anti-metabolite, plant extract, plant alkaloid,nitrosourea, hormone, nucleoside or nucleotide analog; thereby reducingor inhibiting proliferation of the cell.
 3. The method of claim 1,wherein the alkylating agent, anti-metabolite, plant extract, plantalkaloid, nitrosourea, hormone, nucleoside or nucleotide analogcomprises cyclophosphamide, azathioprine, cyclosporin A, prednisolone,melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate,6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside,5-azacytidine (5-AZC), bleomycin, actinomycin D, mithramycin, mitomycinC, carmustine, lomustine, semustine, streptozotocin, hydroxyurea,cisplatin, carboplatin, oxiplatin, mitotane, procarbazine, dacarbazine,taxol, paclitaxel, vinblastine, vincristine, doxorubicin,dibromomannitol, topoisomerase inhibitors, irinotecan, topotecan,etoposide, teniposide, gemcitabine, or pemetrexed.
 4. A method oftreating a subject having a hyperproliferative disorder, the methodcomprising administering: (i) an antibody conjugate comprising anantibody that binds to CD20 or CD19, wherein the antibody is linked totwo or more lytic domains, wherein said lytic domains comprise orconsist of a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF andKFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6, respectively), or a peptideselected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ IDNOs.:1-6, respectively) having one or more of the F, A, or K residues ofthe lytic domain substituted with a corresponding D-amino acid; whereinone of said lytic domains is linked to the amino(NH2)-terminus of aLight (L) chain of the antibody, and the other of said lytic domains islinked to the amino(NH2)-terminus of a Heavy (H) chain of the antibody;and (ii) an alkylating agent, anti-metabolite, plant extract, plantalkaloid, nitrosourea, hormone, nucleoside or nucleotide analog; therebytreating the subject having the hyperproliferative disorder.
 5. A methodof treating a subject having a hyperproliferative disorder, the methodcomprising administering: (i) a polypeptide conjugate comprising a Heavy(H) chain and a Light (L) chain of an antibody that binds to CD20 orCD19, wherein the Heavy (H) and Light (L) chain variable regions eachcomprise 3 CDRs, wherein the amino(NH2)-terminus of the Light (L) chainis linked to a lytic domain, wherein the amino(NH2)-terminus of theHeavy (H) chain is linked to a lytic domain, wherein said lytic domainscomprise or consist of a peptide selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6,respectively), or a peptide selected from KFAKFAKKFAKFAKK,KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA (SEQ ID NOs.:1-6,respectively) having one or more of the F, A, or K residues of the lyticdomain substituted with a corresponding D-amino acid; and (ii) analkylating agent, anti-metabolite, plant extract, plant alkaloid,nitrosourea, hormone, nucleoside or nucleotide analog; thereby treatingthe subject having the hyperproliferative disorder.
 6. The method ofclaim 4, wherein the alkylating agent, anti-metabolite, plant extract,plant alkaloid, nitrosourea, hormone, nucleoside or nucleotide analogcomprises cyclophosphamide, azathioprine, cyclosporin A, prednisolone,melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate,6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside,5-azacytidine (5-AZC), bleomycin, actinomycin D, mithramycin, mitomycinC, carmustine, lomustine, semustine, streptozotocin, hydroxyurea,cisplatin, carboplatin, oxiplatin, mitotane, procarbazine, dacarbazine,taxol, paclitaxel, vinblastine, vincristine, doxorubicin,dibromomannitol, topoisomerase inhibitors, irinotecan, topotecan,etoposide, teniposide, gemcitabine, or pemetrexed.
 7. The method ofclaim 1, wherein the antibody conjugate further comprises a lytic domainlinked to the carboxy(C)-terminus of the Heavy (H) chain.
 8. The methodof claim 1, wherein the antibody conjugate further comprises a lyticdomain linked to the carboxy(C)-terminus of the Light (L) chain.
 9. Themethod of claim 1, wherein the antibody conjugate further comprises aplurality of lytic domains linked to the Heavy (H) chain and Light (L)chain, wherein at least one of the lytic domains is linked to thecarboxy(C)-terminus of the Heavy (H) chain or is linked to thecarboxy(C)-terminus of the Light (L) chain.
 10. The method of claim 1,wherein the antibody conjugate further comprises three, four, five, six,seven or eight lytic domains linked to the Heavy (H) chain or Light (L)chain.
 11. The method of claim 10, wherein the lytic domains are linkedto the amino(NH2)-terminus of the Light (L) chains, and have theidentical amino acid sequence.
 12. The method of claim 1, wherein theantibody conjugate comprising the lytic domain linked to theamino(NH2)-terminus of the Light (L) chain is joined to said Light (L)chain immediately after the last amino acid at the amino(NH2)-terminusof the Light (L) chain, thereby forming a continuous amino acid sequencebetween the lytic domain and the Light (L) chain.
 13. The method ofclaim 1, wherein said Light (L) chain and said lytic domain linked tothe amino(NH2)-terminus of the Light (L) chain are joined by a covalentbond, peptide sequence or a non-peptide linker or spacer.
 14. The methodof claim 13, wherein said linker or spacer comprises a linear carbonchain.
 15. The method of claim 1, wherein said lytic domain consists ofa sequence from about 15 to about 20 L- or D-amino acids, about 15 toabout 28 L- or D-amino acids, about 15 to about 50 L- or D-amino acids.16. The method of claim 1, wherein the lytic domain linked to theamino(NH2)-terminus of the Light (L) chain is cationic.
 17. The methodof claim 1, wherein the lytic domain linked to the amino(NH2)-terminusof the Light (L) chain forms an amphipathic alpha helical structure. 18.The method of claim 1, wherein the antibody conjugate binds to CD20. 19.The method of claim 1, wherein the antibody comprises an antibodyfragment or subsequence that comprises Heavy (H) and Light (L) chainvariable regions each comprising 3 CDRs of an antibody that binds toCD20 or CD19.
 20. The method of claim 19, wherein said antibody fragmentor subsequence comprises an Fab, Fab′, F(ab′)₂, Fv, Fd, single-chain Fv(scFv), disulfide-linked Fvs (sdFv), V_(L), V_(H), Camel Ig, V-NAR, VHH,trispecific (Fab₃), bispecific (Fab₂), diabody ((V_(L)-V_(H))₂ or(V_(H)-V_(L))₂), triabody (trivalent), tetrabody (tetravalent), minibody((scF_(v)-C_(H)3)₂), bispecific single-chain Fv (Bis-scFv),IgGdeltaC_(H)2, scFv-Fc, (scFv)₂-Fc, affibody, aptamer, avimer ornanobody.
 21. The method of claim 1, wherein said antibody is amonoclonal antibody.
 22. The method of claim 1, wherein said Heavy (H)chain or Light (L) chain is selected from a Heavy (H) chain or Light (L)chain of an antibody that binds to CD20 or has all 3 CDR sequences of anantibody that binds to CD20 set forth in any of Table C or Examples 14,15, 18, 20 or
 21. 23. The method of claim 1, wherein said conjugatecomprises any Heavy (H) chain conjugate, any Light (L) chain conjugate,any whole antibody conjugate sequence of an antibody that binds to CD20set forth in any of Table C or Examples 14, 15, 18, 20 or
 21. 24. Themethod of claim 1, wherein said CD19 comprises all or a portion of SEQID NO:132 or wherein said CD20 comprises all or a portion of SEQ IDNO:133.
 25. The method of claim 1, wherein said antibody conjugate isisolated or purified.
 26. The method of claim 1, wherein the methodcomprises treating a subject having a neoplasia, tumor, cancer ormalignancy, comprising administering to the subject an amount of theconjugate sufficient to reduce or inhibit proliferation of theneoplasia, tumor, cancer or malignancy.
 27. The method of claim 10,wherein the lytic domains linked to the amino(NH2)-terminus of the Light(L) chains have a different amino acid sequence.
 28. The method of claim1, wherein the antibody conjugate binds to CD19.